Compositions and methods for the display of proteins on the surface of bacteria and their derived vesicles and uses thereof

ABSTRACT

The present invention relates to compositions and methods for displaying proteins and polypeptides on the surface of cells and cellular vesicles. Methods and compositions for drug and vaccine delivery using cell surface display systems of the present invention are also disclosed.

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/939,506, filed May 22, 2007.

The subject matter of this application was made with support from theUnited States Government under the National Institutes of Health, GrantNo. NIBIB R21EB005669. The U.S. Government has certain rights.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for displayingproteins and polypeptides on the surface of cells and cell vesicles.

BACKGROUND OF THE INVENTION

Protein translocation is a highly conserved process that is essential toall life. Extracellular secretion of virulence factors is a strategyutilized by invading bacteria to establish a colonization niche,communicate with host cells, and modulate host defense and response.With few exceptions, bacterial protein secretion systems arecharacterized by the membrane translocation of a single protein or elsesmall protein complexes (Christie et al., “Bacterial Type IV Secretion:Conjugation Systems Adapted to Deliver Effector Molecules to HostCells,” Trends Microbiol 8:354-60 (2000); Galan et al., “Type IIISecretion Machines: Bacterial Devices for Protein Delivery into HostCells,” Science 284:1322-8 (1999); Gentschev et al., “The E. colialpha-Hemolysin Secretion System and its Use in Vaccine Development,”Trends Microbiol 10:39-45 (2002); Henderson et al., “AutotransporterProteins, Evolution and Redefining Protein Secretion,” Trends Microbiol8:529-32 (2000); and Russel M., “Macromolecular Assembly and SecretionAcross the Bacterial Cell Envelope: Type II Protein Secretion Systems,”J Mol Biol 279:485-99 (1998)). Recently, however, the production andrelease of outer membrane vesicles (OMVs) has been demonstrated as anovel secretion mechanism for the transmission of a diverse group ofproteins and lipids to mammalian cells (Kuehn M. J. et al., “BacterialOuter Membrane Vesicles and the Host-Pathogen Interaction,” Genes Dev19:2645-55 (2005)). OMVs are small proteoliposomes with an averagediameter of 50-200 nm that are constitutively released from the outermembrane of pathogenic and non-pathogenic species of Gram-negativebacteria during growth (Beveridge T. J., “Structures of Gram-NegativeCell Walls and their Derived Membrane Vesicles,” J Bacteriol 181:4725-33(1999)). Biochemical analysis has demonstrated that OMVs are comprisedof outer membrane proteins, lipopolysaccharide, phospholipids andsoluble periplasmic proteins, (Horstman et al., “EnterotoxigenicEscherichia coli Secretes Active Heat-Labile Enterotoxin Via OuterMembrane Vesicles,” J Biol Chem 275:12489-96 (2000) and McBroom et al.,“Outer Membrane Vesicles,” In EcoSal—Escherichia coli and Salmonella:Cellular and Molecular Biology (III, R. C., ed.). ASM Press, Washington,D.C. (2005)) the latter of which become trapped in the vesicle lumenduring release from the cell surface. OMVs are largely devoid of innermembrane and cytoplasm components although several studies indicate thatchromosomal, phage and plasmid DNA can infiltrate OMVs as a means ofOMV-mediated transfer of genetic information between bacteria (Dorwardet al., “Export and Intercellular Transfer of DNA Via Membrane Blebs ofNeisseria gonorrhoeae,” J Bacteriol 171:2499-505 (1989); Kolling et al.,“Export of Virulence Genes and Shiga Toxin by Membrane Vesicles ofEscherichia coli O157:H7,” Appl Environ Microbiol 65:1843-8 (1999);Yaron et al., “Vesicle-Mediated Transfer of Virulence Genes fromEscherichia coli O157:H7 to Other Enteric Bacteria,” Appl EnvironMicrobiol 66:4414-20 (2000); and Renelli et al., “DNA-ContainingMembrane Vesicles of Pseudomonas aeruginosa PAO1 and their GeneticTransformation Potential,” Microbiology 150:2161-9 (2004)).

An intriguing yet poorly understood phenomena pertaining to OMVs is theobservation that certain membrane and/or soluble periplasmic proteinsare enriched in vesicles while others are preferentially excluded. Themajority of these enriched proteins happen to be virulence factorsincluding, for example, Escherichia coli cytolysin A (ClyA), (Wai etal., “Vesicle-Mediated Export and Assembly of Pore-Forming Oligomers ofthe Enterobacterial ClyA Cytotoxin,” Cell 115:25-35 (2003))enterotoxigenic E. coli heat labile enterotoxin (LT), (Horstman et al.,“Enterotoxigenic Escherichia coli Secretes Active Heat-LabileEnterotoxin Via Outer Membrane Vesicles,” J Biol Chem 275:12489-96(2000)) and Actinobacillus actinomycetemcomitans leukotoxin, (Kato etal., “Outer Membrane-Like Vesicles Secreted by Actinobacillusactinomycetemcomitans are Enriched in Leukotoxin,” Microb Pathog32:1-13. (2002)) whereas proteins that are excluded from OMVs includenumerous unidentified outer membrane (OM) proteins (Kato et al., “OuterMembrane-Like Vesicles Secreted by Actinobacillus actinomycetemcomitansare Enriched in Leukotoxin,” Microb Pathog 32:1-13. (2002)) as well asE. coli DsbA (Wai et al., “Vesicle-Mediated Export and Assembly ofPore-Forming Oligomers of the Enterobacterial ClyA Cytotoxin,” Cell115:25-35 (2003)). The preferential exclusion of proteins raises theinteresting possibility that a yet-to-be determined sorting mechanismexists in the bacterial periplasm for discriminatory loading of a highlyspecific subset of proteins into OMVs (Wai et al., “Vesicle-MediatedExport and Assembly of Pore-Forming Oligomers of the EnterobacterialClyA Cytotoxin,” Cell 115:25-35 (2003) and McBroom et al., “Release ofOuter Membrane Vesicles by Gram-Negative Bacteria is a Novel EnvelopeStress Response,” Mol Microbiol 63:545-58 (2007)). Moreover, theobservation that certain virulence factors are enriched in vesiclessuggests that OMVs may play a key role in bacterial pathogenesis bymediating transmission of active virulence factors and other bacterialenvelope components to host cells. Indeed, numerous vesicle-associatedvirulence factors (e.g., adhesins, immunomodulatory compounds, proteasesand toxins) have been shown to induce cytotoxicity, confer vesiclebinding to and invasion of host cells, and modulate the host immuneresponse (Horstman et al., “Enterotoxigenic Escherichia coli SecretesActive Heat-Labile Enterotoxin Via Outer Membrane Vesicles,” J Biol Chem275:12489-96 (2000); Fiocca et al., “Release of Helicobacter pyloriVacuolating Cytotoxin by Both a Specific Secretion Pathway and Buddingof Outer Membrane Vesicles. Uptake of Released Toxin and Vesicles byGastric Epithelium,” J Pathol 188:220-6 (1999); Keenan et al., “A Rolefor the Bacterial Outer Membrane in the Pathogenesis of Helicobacterpylori Infection,” FEMS Microbiol Lett 182:259-64 (2000); Kadurugamuwaet al., “Delivery of the Non-Membrane-Permeative Antibiotic Gentamicininto Mammalian Cells by Using Shigella flexneri Membrane Vesicles,”Antimicrob Agents Chemother 42:1476-83 (1998); and Kesty et al.,“Enterotoxigenic Escherichia coli Vesicles Target Toxin Delivery intoMammalian Cells,” EMBO J. 23:4538-49 (2004)).

To date, one of the best studied vesicle-associated virulence factors isthe 34-kDa cytotoxin ClyA (also called HlyE or SheA) found in pathogenicand non-pathogenic E. coli strains (Wai et al., “Vesicle-Mediated Exportand Assembly of Pore-Forming Oligomers of the Enterobacterial ClyACytotoxin,” Cell 115:25-35 (2003) and del Castillo et al., “TheEscherichia coli K-12 SheA Gene Encodes a 34-kDa Secreted Haemolysin,”Mol Microbiol 25:107-15 (1997)) and also in Salmonella enterica serovarsTyphi and Paratyphi A (Oscarsson et al., “Characterization of aPore-Forming Cytotoxin Expressed by Salmonella enterica serovars typhiand paratyphi A,” Infect Immun 70:5759-69 (2002)). Structural studiesindicate that the water-soluble form of ClyA is a bundle of four majorα-helices, with a small surface-exposed hydrophobic beta-hairpin at the“head” end of the structure, and the N- and C-termini at the “tail” end(Wallace et al., “E. coli Hemolysin E (HlyE, ClyA, SheA): X-ray CrystalStructure of the Toxin and Observation of Membrane Pores by ElectronMicroscopy,” Cell 100:265-76 (2000)) while lipid-associated ClyA formsan oligomeric pore complex comprised of either 8 or 13 ClyA subunits(Eifler et al., “Cytotoxin ClyA from Escherichia coli Assembles to a13-meric Pore Independent of its Redox-State,” EMBO J25:2652-61 (2006)and Tzokov et al., “Structure of the Hemolysin E (HlyE, ClyA, SheA)Channel in its Membrane-Bound Form,” J Biol Chem 281:23042-9 (2006)).Expression of the clyA gene is silenced in non-pathogenic E. coli K-12laboratory strains by the nucleoid protein H-NS (Westermark et al.,“Silencing and Activation of ClyA Cytotoxin Expression in Escherichiacoli,” J Bacteriol 182:6347-57 (2000)) but is derepressed inH-NS-deficient E. coli, thereby inducing cytotoxicity towards culturedmammalian cells (Gomez-Gomez et al., “Hns Mutant Unveils the Presence ofa Latent Haemolytic Activity in Escherichia coli K-12,” Mol Microbiol19:909-10 (1996)). More recent evidence indicates that ClyA is exportedfrom E. coli in OMVs and retains a cytolytically active, oligomericconformation in the vesicles (Wai et al., “Vesicle-Mediated Export andAssembly of Pore-Forming Oligomers of the Enterobacterial ClyACytotoxin,” Cell 115:25-35 (2003)). However, the route by which ClyAmanages to cross the bacterial IM and assemble in OMVs remains amystery, as it carries no canonical signal peptide (del Castillo et al.,“The Escherichia coli K-12 SheA Gene Encodes a 34-kDa SecretedHaemolysin,” Mol Microbiol 25:107-15 (1997)) and is not N-terminallyprocessed (Ludwig et al., “Analysis of the SlyA-Controlled Expression,Subcellular Localization and Pore-Forming Activity of a 34 kDaHaemolysin (ClyA) from Escherichia coli K-12,” Mol Microbiol 31:557-67(1999)). Also undetermined is the role that ClyA plays invesicle-mediated interactions with mammalian cells.

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

A first aspect of the present invention is directed to a method ofdisplaying a protein on a cell surface. This method involves providingeither a fusion protein containing at least a portion of a ClyA proteinand at least a portion of a second protein coupled to said ClyA proteinor a nucleic acid construct encoding the fusion protein. The fusionprotein or the nucleic acid construct is administered to a cell underconditions effective to display the fusion protein on the surface of thecell.

The present invention is also directed to a cell displaying a ClyAfusion protein, where the ClyA fusion protein comprises at least aportion of a ClyA protein and at least a portion of a second proteincoupled to the ClyA protein.

Another aspect of the present invention relates to a method ofdisplaying a protein on cell vesicles. This method involves providingeither a fusion protein containing at least a portion of a ClyA proteinand at least a portion of a second protein coupled to said ClyA proteinor a nucleic acid construct encoding the fusion protein. The fusionprotein or the nucleic acid construct is administered to a cell underconditions effective to display the fusion protein on the vesicles ofthe cell.

The present invention is also directed to a vesicle displaying a ClyAfusion protein, where the ClyA fusion protein comprises at least aportion of a ClyA protein and at least a portion of a second proteincoupled to the ClyA protein.

Another aspect of the present invention is directed to a method ofimaging cells, which involves providing either a fusion proteincomprising at least a portion of a ClyA protein and marker proteincoupled to the ClyA protein, or a nucleic acid construct encoding thefusion protein. The method further involves administering to a cell thefusion protein or the nucleic acid construct under conditions effectiveto display the fusion protein on the cell and imaging the cell based onthe presence of the marker protein.

Another aspect of the present invention is directed to a method ofsorting cells, which involves providing either a fusion proteincomprising at least a portion of a ClyA protein and marker proteincoupled to the ClyA protein, or a nucleic acid construct encoding thefusion protein. The method further involves administering to a cell thefusion protein or the nucleic acid construct under conditions effectiveto display the fusion protein on the cell and sorting the cell based onthe presence of the marker protein.

The present invention is also directed to a method of screening alibrary of candidate compounds to identify compounds that bind to atarget protein. This method involves providing the library of candidatecompounds to be screened and a cell or a cell vesicle displaying a ClyAfusion protein. The ClyA fusion protein comprises at least a portion ofthe ClyA protein and at least a portion of a second protein, where thesecond protein of the ClyA fusion protein comprises the target protein.The method further includes contacting the library of candidatecompounds with the cell or cell vesicle displaying the ClyA fusiontarget protein under conditions effective for the candidate compound tobind to the target protein and identifying those compounds that bind tothe target protein.

Another aspect of the present invention relates to a method ofdelivering a therapeutic agent to a cell, which involves providing avesicle displaying a ClyA fusion protein, where the ClyA fusion proteincomprises at least a portion of the ClyA protein and at least a portionof a second protein. The vesicle contains the therapeutic agent to bedelivered and the second protein of the ClyA fusion protein comprises atargeting protein. The vesicle is administered to a cell underconditions effective to deliver the therapeutic agent to the cell.

The present invention is also directed to a method of eliciting animmune response in a mammal. This method involves providing a cell or acell vesicle displaying a ClyA fusion protein. The ClyA fusion proteincomprises at least a portion of the ClyA protein and at least a portionof a second protein, where the second protein of the ClyA fusion proteincomprises an antigenic protein or peptide capable of eliciting an immuneresponse in the mammal. The cell or vesicle is administered to themammal under conditions effective to elicit the immune response.

The present invention is also directed to drug and vaccine deliveryvehicles consisting of a cell vesicles displaying a ClyA fusion protein.The ClyA fusion protein comprises at least a portion of the ClyA proteinand at least a portion of a second protein.

The present invention describes the engineering of synthetic membranevesicles (s-MVs) with non-native functions that are useful for a widerange of applications including, for instance, the analysis of thecomplete ClyA translocation process. Specifically, s-MVs have beenprogrammed with enhanced functionality by creating chimeras betweenheterologous proteins such as green fluorescent protein (GFP) orβ-lactamase (Bla) and ClyA. Using these engineered vesicles, it has beendetermined that ClyA is capable of co-localizing a variety ofstructurally diverse fusion partners to the surface of E. coli and theirreleased vesicles, but only when the periplasmic disulfide bond-formingmachinery was present. Importantly, these cell- and OMV-associatedproteins retained their biological activity, suggesting that thefunctionality of natural OMVs can be easily expanded via the expressionof ClyA chimeras.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-F show the subcellular localization of ClyA and ClyA fusionproteins. FIG. 1A is an electron micrograph of vesicles derived fromJC8031 cells expressing ClyA-GFP. Bar is equal to 100 nm. FIG. 1B is agraph showing the Z-average particle size of 1 ml vesicle suspensionscontaining ˜30 μg/ml total protein obtained from plasmid-free orClyA-GFP-expressing JC8031 cells. Error bars represent the standarddeviation of 3 replicates. FIG. 1C is a Western blot of vesiclefractions isolated from E. coli strain JC8031 expressing GFP, ClyA-GFPand GFP-ClyA. The blot was probed with anti-GFP serum. FIG. 1D is aWestern blot showing protein detection and FIG. 1E is a graph of GFPfluorescence detection in periplasmic (per), cytoplasmic (cyt) andvesicle (OMV) fractions generated from JC8031 or BW25113 nlpI::Kan cellsexpressing ClyA-His6, ClyA-GFP, and GFP-ClyA. The ClyA-His6 blot wasfirst probed with anti-polyhistidine. ClyA-GFP and GFP-ClyA blots wereprobed with anti-GFP. Following stripping of membranes, blots werereprobed with anti-OmpA serum or anti-DsbA serum as indicated. Allfractions were generated from an equivalent number of cells. FIG. 1F isfluorescence microscopy images of vesicles generated from JC8031 cellsexpressing ClyA-His6, ClyA-GFP, and GFP-ClyA.

FIGS. 2A-C show the density gradient fractionation of vesicles. FIG. 2Ais a electrophoretic analysis of the density gradient fractions from top(lane 1, lowest density) to bottom (lane 10, highest density) fromJC8031 cells expressing ClyA-GFP. The bands corresponding to ClyA-GFP(top), OmpA (middle) and DsbA (bottom) were obtained with anti-GFP,anti-OmpA and anti-DsbA serum, respectively. Lane (i) represents inputvesicles from the purified cell-free supernatant. FIG. 2B shows thequantification of the ClyA-GFP levels (filled squared) in each fractionas determined by densitometry using ImageJ software. Band intensityvalues were normalized to the maximum intensity corresponding to theClyA-GFP measured in fraction 7. Also plotted is the GFP activity (opensquares) measured in each fraction and normalized to the maximumactivity, which also corresponds to fraction 7. FIG. 2C is fluorescencemicroscopy images of vesicles that migrated in gradient fractions 7 and10.

FIGS. 3A-B depict the microscopic analysis of ClyA expression. JC8031cells grown at 37° C. in LB were induced to express GFP (FIG. 3A) andClyA-GFP (FIG. 3B). For immunofluorescence microscopy, cells weretreated with mouse monoclonal anti-GFP and subsequently withrhodamine-conjugated anti-mouse IgG. Panels show phase contrastmicroscopy and fluorescence microscopy using green and red emissionfilters as indicated. For immunoelectron microscopy, cells were treatedwith mouse monoclonal anti-GFP and subsequently with gold-conjugatedanti-mouse IgG. Arrows indicate the 25 nm gold particles. The bars areequal to 500 nm.

FIGS. 4A-D depicts the detection of vesicles and vesicle-associatedantigens via immuno-Surface Plasmon Resonance (SPR). FIG. 4A is afluorescence microscopy analysis of the binding of fluorescent s-MVs (ata concentration of 0.35 μg/μl) to anti-E. coli antibody in the testchannel and to the BSA-treated surface in the reference channelfollowing 20 min of s-MV binding and 20 min PBS rinse. The measurementsrepresent the size of the polydimethylsiloxane (PDMS) master. FIG. 4B isan overlay of SPR sensorgrams showing concentration-dependent binding ofOMVs to immobilized anti-E. coli antibodies. For each bindingexperiment, 200 μl of OMV-containing samples (diluted to theconcentrations indicated) were introduced to the test or referencechannel for 20 min, followed by a 20 min PBS rinse. The SPR signal wasrecorded as wavelength shift (nm) versus time and plotted as a“sensorgram”. All binding experiments were performed at 25° C.±1° C.with a flowrate of 10 μl/min. Each vesicle sample was assayed intriplicate and the standard error was determined to be less than 5%.FIG. 4C shows the vesicle standard curve generated using the SPRimmunosensor. The steady-state SPR signal change was calculated bysubtracting the average SPR signal during the PBS wash step followingOMV binding from the average SPR signal collected during the initial PBSwash step prior to OMV addition. The equation y=0.92ln(x)+3.63 with anR² value of 0.95 describes the fit of a straight line through thelogarithm of the data and was determined using SigmaPlot. The resultsare the average values calculated for the SPR signal change from threeindependent binding measurements with error bars showing ±standarderror. It is noteworthy that the lower detection limit for this systemwas determined to be 0.01 μg/μl (10% of the compensated SPR wavelengthshift in the standard curve) and that for vesicle concentrations ≧0.18μg/μl, SPR wavelength shifts >2.5 nm were recorded, which is ˜10×greater than the baseline signal. FIG. 4D shows representativesensorgrams for antibody binding to s-MV surface-displayed GFP in testand reference channels. Channels were prepared identically so thatfluorescent s-MVs were captured in both channels. The change in SPRsignal over time was measured following addition of 1 μg/μl anti-GFP(black line) or 1 μg/μl anti-his6x (gray line) monoclonal antibody tosurface-captured s-MVs. Antibody binding proceeded for 20 min followedby a 10 min PBS rinse. Each antibody was assayed in triplicate and thestandard error was determined to be less than 5%.

FIGS. 5A-D illustrate the biochemical and genetic analysis of ClyAlocalization. Proteinase K susceptibility of OMV-tethered GFP wasdetermined by fluorescence microscopy of vesicles generated from JC8031cells expressing ClyA-GFP treated with Proteinase K and SDS as indicated(FIG. 5A) and Western blot of vesicles generated from JC8031 cellsexpressing ClyA-GFP or GFP-ClyA (FIG. 5B). Blots were probed with mouseanti-GFP (left panels) or anti-ClyA serum (right panels). The molecularweight (MW) ladder is marked at left. An equivalent number of vesicleswas used in all cases. FIG. 5C is a Western blot analysis of periplasmicand OMV fractions from JC8031 (dsbA+) and JC8031 dsbA::Kan cells (dsbA−)expressing either ClyA-GFP or ClyA-His6 as indicated. FIG. 5D shows theimmunofluorescence of wt JC8031 (dsbA+, top left and center panels) andJC8031 dsbA::Kan cells (dsbA−, bottom left and center panel) expressingpClyA-GFP or pClyA-His6 as indicated and fluorescence of vesicles (rightpanels) derived from the same cells as indicated. Forimmunofluorescence, cells were treated with either mouse monoclonalanti-GFP or anti-polyhistidine antibodies and subsequently withrhodamine-conjugated anti-mouse IgG.

FIGS. 6A-D show the interaction of ClyA-GFP vesicles with HeLa cells.FIG. 6A is fluorescent images of OMVs containing ClyA-GFP that wereincubated with HeLa cells at 37° C. for 30 min or 3 hours as indicated.Fixed cells were stained with 0.5 mg/mL ethidium bromide (EtBr, upperpanels) and visualized by fluorescence microscopy. FIG. 6B arefluorescent images showing the temperature dependence of OMV-HeLa cellinteractions. This interaction was examined by incubation of HeLa cellswith GFP-ClyA OMVs at the temperatures indicated. An equivalent numberof OMVs (˜150 μg) was used in all cases. FIG. 6C is fluorescent imagescomparing untreated (−G_(M1)) or pretreated (+G_(M1)) OMVs from JC8031cells expressing ClyA-GFP that were incubated with HeLa cells at 37° C.for 3 hours. Fixed cells were stained with 0.5 mg/mL ethidium bromide(EtBr, upper panels) and visualized by fluorescence microscopy. Anequivalent number of OMVs (˜150 μg) was used in all cases. FIG. 6D showsthe cytotoxicity of vesicles as measured using the MTS assay with HeLacell cultures. Percent viability is reported as the viability ofvesicle-treated HeLa cells normalized to the viability followingtreatment with PBS. HeLa cells were treated with vesicle solutionsderived from plasmid-free JC8031 cells and JC8031 cells expressingClyA-His6, ClyA-GFP. An equivalent number of OMVs (˜150 μg) was used inall cases. Each sample was assayed in triplicate with error bars showing±standard error.

FIGS. 7A-B show the creation of immuno-MVs via ClyA-scFv chimeras. FIG.7A is fluorescence microscopy images of whole cells and vesiclesgenerated from JC8031 cells expressing scFv.Dig, ClyA-scFv.Dig orLpp-OmpA-scFv.Dig as indicated. For these studies, cells were grown andinduced at room temperature followed by fluorescent labeling of cells ortheir derived vesicles with 1 μM Dig-BODIPY for 1 h at room temperature.FIG. 7B shows the genetic analysis of scFv.Dig localization, performedusing flow cytometric analysis of strains and plasmids as indicated.Cells were grown and induced at room temperature followed by labelingwith 1 μM Dig-BODIPY for 1 h at room temperature. Fluorescence isreported as the mean fluorescence for each cell population and wasassayed in triplicate with error bars showing ±standard error.

FIGS. 8A-C show that the fusion of GFP to the C-terminus of ClyA resultsin expression of a 61 kDa chimeric protein that exhibits fluorescenceand hemolytic activity. FIG. 8A is a Western blot of purifiedhis6-tagged recombinant proteins with anti-polyHistidine IgG. Theexpected molecular weights for ClyA, GFP, and ClyA-GFP fusion are 27kDa, 34 kDa, and 61 kDa, respectively. FIG. 8B provides the relativehemolysis activity of ClyA, ClyA-GFP, and GFP. The intrinsic hemolysisactivity of ClyA is retained in the ClyA-GFP fusion protein, andincreases with increasing concentration. FIG. 8C shows the fluorescenceintensity of GFP and recombinant ClyA-GFP in relative fluorescence units(RFU, arbitrary units). The fluorescence intensity of ClyA-GFP and GFPincreases linearly with increasing concentration.

FIGS. 9A-E depict the characterization of recombinant outer membranevesicles from the E. coli vesicle hyperproducing strain, JC8031,expressing ClyA-GFP. FIG. 9A is an electron micrograph of empty OMVsstained by uranyl acetate. The scale bar represents 200 nm. This imageis also representative of recombinant OMV containing ClyA-GFP. FIG. 9Bis a fluorescence micrograph of ClyA-GFP in association with recombinantOMV. FIG. 9C is a Western blot with anti-GFP antibodies of cell-freeculture supernatants and OMV suspensions from cultures of E. coliexpressing the empty plasmid vector or ClyA-GFP. FIG. 9D shows theZ-average hydrodynamic diameter of empty and recombinant OMV suspensionsin PBS, as measured by dynamic light scattering. FIG. 9E shows thelipopolysaccharide content in empty and recombinant OMV suspensions,normalized by total protein content. The asterisk (*) denotesstatistical significance (p<0.05), as determined by the student'st-test.

FIGS. 10A-D illustrate the significantly enhanced immunogenicity of GFPwhen fused with ClyA. Individual host anti-GFP IgG responses in serumdiluted 1:12,800. Groups of five BALB/c mice were subcutaneouslyimmunized with: 2.5 μg GFP (group I/FIG. 10A), 2.5 μg ClyA (groupII/FIG. 10B), 5 μg ClyA-GFP fusion (group III/FIG. 10), and 2.5 μg ClyAmixed with 2.5 μg GFP (group IV/FIG. 10D). Mice were immunized on day 0and day 28, as marked by the arrowheads in each chart. The daggers (†)represent statistical significance (p<0.05) of antibody titers in groupIII compared to titers in groups II and IV.

FIGS. 11A-B demonstrate ClyA-GFP administered in recombinant OMV retainsits immunogenicity in mice. Individual host anti-GFP IgG titers in serumdiluted 1:12,800. Groups of five BALB/c mice were immunized withpurified ClyA-GFP fusion mixed with empty OMV (group V/FIG. 11A), andClyA-GFP fusion in association with recombinant OMV (group VI/FIG. 11B).The effective dose of ClyA-GFP in both treatment groups was 2.5 μg. Micewere immunized on day 0 and day 28, as marked by the arrowheads in eachchart. The asterisks (*) denote statistically significant difference(p<0.05) in comparison with antibody titers in the purified ClyA-GFPtreatment group (group III/FIG. 10C) on the corresponding day.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention is directed to a method ofdisplaying a protein on a cell surface. This method involves providingeither a fusion protein containing at least a portion of a ClyA proteinand at least a portion of a second protein coupled to said ClyA proteinor a nucleic acid construct encoding the fusion protein. The fusionprotein or the nucleic acid construct is administered to a cell underconditions effective to display the fusion protein on the surface of thecell.

The present invention is also directed to a cell displaying a ClyAfusion protein, where the ClyA fusion protein comprises at least aportion of a ClyA protein and at least a portion of a second proteincoupled to the ClyA protein.

Another aspect of the present invention relates to a method ofdisplaying a protein on cell vesicles. This method involves providingeither a fusion protein containing at least a portion of a ClyA proteinand at least a portion of a second protein coupled to said ClyA proteinor a nucleic acid construct encoding the fusion protein. The fusionprotein or the nucleic acid construct is administered to a cell underconditions effective to display the fusion protein on the vesicles ofthe cell.

The present invention is also directed to a vesicle displaying a ClyAfusion protein, where the ClyA fusion protein comprises at least aportion of a ClyA protein and at least a portion of a second proteincoupled to the ClyA protein.

The ClyA fusion protein used in accordance with the methods andcompositions of the present invention may comprise either a full lengthor polypeptide fragment, analogue or derivative thereof of the ClyAprotein from E. coli (Genbank Accession No. AJ001829). The amino acidsequence of the E. coli ClyA protein is set forth below as SEQ ID NO: 1:

Met Thr Glu Ile Val Ala Asp Lys Thr Val Glu Val Val Lys Asn Ala1               5                   10                  15 Ile Glu ThrAla Asp Gly Ala Leu Asp Leu Tyr Asn Lys Tyr Leu Asp            20                  25                  30 Gln Val Ile ProTrp Gln Thr Phe Asp Glu Thr Ile Lys Glu Leu Ser        35                  40                  45 Arg Phe Lys Gln GluTyr Ser Gln Ala Ala Ser Val Leu Val Gly Asp    50                  55                  60 Ile Lys Thr Leu Leu MetAsp Ser Gln Asp Lys Tyr Phe Glu Ala Thr65                  70                  75                  80 Gln ThrVal Tyr Glu Trp Cys Gly Val Ala Thr Gln Leu Leu Ala Ala                85                  90                  95 Tyr Ile LeuLeu Phe Asp Glu Tyr Asn Glu Lys Lys Ala Ser Ala Gln            100                 105                 110 Lys Asp Ile LeuIle Lys Val Leu Asp Asp Gly Ile Thr Lys Leu Asn        115                 120                 125 Glu Ala Gln Lys SerLeu Leu Val Ser Ser Gln Ser Phe Asn Asn Ala    130                 135                 140 Ser Gly Lys Leu Leu AlaLeu Asp Ser Gln Leu Thr Asn Asp Phe Ser145                 150                 155                 160 Glu LysSer Ser Tyr Phe Gln Ser Gln Val Asp Lys Ile Arg Lys Glu                165                 170                 175 Ala Tyr AlaGly Ala Ala Ala Gly Val Val Ala Gly Pro Phe Gly Leu            180                 185                 190 Ile Ile Ser TyrSer Ile Ala Ala Gly Val Val Glu Gly Lys Leu Ile        195                 200                 205 Pro Glu Leu Lys AsnLys Leu Lys Ser Val Gln Asn Phe Phe Thr Thr    210                 215                 220 Leu Ser Asn Thr Val LysGln Ala Asn Lys Asp Ile Asp Ala Ala Lys225                 230                 235                 240 Leu LysLeu Thr Thr Glu Ile Ala Ala Ile Gly Glu Ile Lys Thr Glu                245                 250                 255 Thr Glu ThrThr Arg Phe Tyr Val Asp Tyr Asp Asp Leu Met Leu Ser            260                 265                 270 Leu Leu Lys GluAla Ala Lys Lys Met Ile Asn Thr Cys Asn Glu Tyr        275                 280                 285 Gln Lys Arg His GlyLys Lys Thr Leu Phe Glu Val Pro Glu Val    290                 295                 300

The E. coli ClyA protein is encoded by the nucleic acid sequence of SEQID NO: 2:

agaaataaag acattgacgc atcccgcccg gctaactatg aattagatga agtaaaattt 60attaatagtt gtaaaacagg agtttcatta caatttatat atttaaagag gcgaatgatt 120atgactgaaa tcgttgcaga taaaacggta gaagtagtta aaaacgcaat cgaaaccgca 180gatggagcat tagatcttta taataaatat ctcgatcagg tcatcccctg gcagaccttt 240gatgaaacca taaaagagtt aagtcgcttt aaacaggagt attcacaggc agcctccgtt 300ttagtcggcg atattaaaac cttacttatg gatagccagg ataagtattt tgaagcaacc 360caaacagtgt atgaatggtg tggtgttgcg acgcaattgc tcgcagcgta tattttgcta 420tttgatgagt acaatgagaa gaaagcatcc gcccagaaag acattctcat taaggtactg 480gatgacggca tcacgaagct gaatgaagcg caaaaatccc tgctggtaag ctcacaaagt 540ttcaacaacg cttccgggaa actgctggcg ttagatagcc agttaaccaa tgatttttca 600gaaaaaagca gctatttcca gtcacaggta gataaaatca ggaaggaagc atatgccggt 660gccgcagccg gtgtcgtcgc cggtccattt ggattaatca tttcctattc tattgctgcg 720ggcgtagttg aaggaaaact gattccagaa ttgaagaaca agttaaaatc tgtgcagaat 780ttctttacca ccctgtctaa cacggttaaa caagcgaata aagatatcga tgccgccaaa 840ttgaaattaa ccaccgaaat agccgccatc ggtgagataa aaacggaaac tgaaacaacc 900agattctacg ttgattatga tgatttaatg ctttctttgc taaaagaagc ggccaaaaaa 960atgattaaca cctgtaatga gtatcagaaa agacacggta aaaagacact ctttgaggta 1020cctgaagtct gataagcgat tattctctcc atgtactcaa ggtataaggt ttatcacatt 1080

In another embodiment of the present invention, the fusion proteincomprises either a full length or polypeptide fragment, analogue orderivative thereof of the ClyA protein derived from Salmonella entericaserovar Typhi (Genbank Accession No. AJ313034). The amino acid sequenceof the S. enterica Typhi ClyA protein is set forth below as SEQ ID NO:3:

Met Thr Gly Ile Phe Ala Glu Gln Thr Val Glu Val Val Lys Ser Ala1               5                   10                  15 Ile Glu ThrAla Asp Gly Ala Leu Asp Leu Tyr Asn Lys Tyr Leu Asp            20                  25                  30 Gln Val Ile ProTrp Lys Thr Phe Asp Glu Thr Ile Lys Glu Leu Ser        35                  40                  45 Arg Phe Lys Gln GluTyr Ser Gln Glu Ala Ser Val Leu Val Gly Asp    50                  55                  60 Ile Lys Val Leu Leu MetAsp Ser Gln Asp Lys Tyr Phe Glu Ala Thr65                  70                  75                  80 Gln ThrVal Tyr Glu Trp Cys Gly Val Val Thr Gln Leu Leu Ser Ala                85                  90                  95 Tyr Ile LeuLeu Phe Asp Glu Tyr Asn Glu Lys Lys Ala Ser Ala Gln            100                 105                 110 Lys Asp Ile LeuIle Arg Ile Leu Asp Asp Gly Val Lys Lys Leu Asn        115                 120                 125 Glu Ala Gln Lys SerLeu Leu Thr Ser Ser Gln Ser Phe Asn Asn Ala    130                 135                 140 Ser Gly Lys Leu Leu AlaLeu Asp Ser Gln Leu Thr Asn Asp Phe Ser145                 150                 155                 160 Glu LysSer Ser Tyr Phe Gln Ser Gln Val Asp Arg Ile Arg Lys Glu                165                 170                 175 Ala Tyr AlaGly Ala Ala Ala Gly Ile Val Ala Gly Pro Phe Gly Leu            180                 185                 190 Ile Ile Ser TyrSer Ile Ala Ala Gly Val Ile Glu Gly Lys Leu Ile        195                 200                 205 Pro Glu Leu Asn AsnArg Leu Lys Thr Val Gln Asn Phe Phe Thr Ser    210                 215                 220 Leu Ser Ala Thr Val LysGln Ala Asn Lys Asp Ile Asp Ala Ala Lys225                 230                 235                 240 Leu LysLeu Ala Thr Glu Ile Ala Ala Ile Gly Glu Ile Lys Thr Glu                245                 250                 255 Thr Glu ThrThr Arg Phe Tyr Val Asp Tyr Asp Asp Leu Met Leu Ser            260                 265                 270 Leu Leu Lys GlyAla Ala Lys Lys Met Ile Asn Thr Cys Asn Glu Tyr        275                 280                 285 Gln Gln Arg His GlyLys Lys Thr Leu Phe Glu Val Pro Asp Val    290                 295                 300

The S. enterica Typhi ClyA protein is encoded by the nucleic acidsequence of SEQ ID NO: 4:

ggaggtaata ggtaagaata ctttataaaa caggtactta attgcaattt atatatttaa 60agaggcaaat gattatgacc ggaatatttg cagaacaaac tgtagaggta gttaaaagcg 120cgatcgaaac cgcagatggg gcattagatc tttataacaa atacctcgac caggtcatcc 180cctggaagac ctttgatgaa accataaaag agttaagccg ttttaaacag gagtactcgc 240aggaagcttc tgttttagtt ggtgatatta aagttttgct tatggacagc caggacaagt 300attttgaagc gacacaaact gtttatgaat ggtgtggtgt cgtgacgcaa ttactctcag 360cgtatatttt actatttgat gaatataatg agaaaaaagc atcagcccag aaagacattc 420tcattaggat attagatgat ggtgtcaaga aactgaatga agcgcaaaaa tctctcctga 480caagttcaca aagtttcaac aacgcttccg gaaaactgct ggcattagat agccagttaa 540ctaatgattt ttcggaaaaa agtagttatt tccagtcaca ggtggataga attcgtaagg 600aagcttatgc cggtgctgca gccggcatag tcgccggtcc gtttggatta attatttcct 660attctattgc tgcgggcgtg attgaaggga aattgattcc agaattgaat aacaggctaa 720aaacagtgca aaatttcttt actagcttat cagctacagt gaaacaagcg aataaagata 780tcgatgcggc aaaattgaaa ttagccactg aaatagcagc aattggggag ataaaaacgg 840aaaccgaaac aaccagattc tacgttgatt atgatgattt aatgctttct ttattaaaag 900gagctgcaaa gaaaatgatt aacacctgta atgaatacca acaaagacac ggtaagaaga 960cgcttttcga ggttcctgac gtctgataca ttttcattcg atctgtgtac ttttaacgcc 1020cgatagcgta aagaaaatga gagacggaga aaaagcgata ttcaacagcc cgataaacaa 1080gagtcgttac cgggctgacg ag 1102

In a further embodiment of the present invention, the fusion proteincomprises either a full length or polypeptide fragment, analogue orderivative thereof of the ClyA protein from Salmonella paratyphi(Genbank Accession No. AJ313033). The amino acid sequence of the S.paratyphi ClyA protein is set forth below as SEQ ID NO: 5:

Met Thr Gly Ile Phe Ala Glu Gln Thr Val Glu Val Val Lys Ser Ala1               5                   10                  15 Ile Glu ThrAla Asp Gly Ala Leu Asp Phe Tyr Asn Lys Tyr Leu Asp            20                  25                  30 Gln Val Ile ProTrp Lys Thr Phe Asp Glu Thr Ile Lys Glu Leu Ser        35                  40                  45 Arg Phe Lys Gln GluTyr Ser Gln Glu Ala Ser Val Leu Val Gly Asp    50                  55                  60 Ile Lys Val Leu Leu MetAsp Ser Gln Asp Lys Tyr Phe Glu Ala Thr65                  70                  75                  80 Gln ThrVal Tyr Glu Trp Cys Gly Val Val Thr Gln Leu Leu Ser Ala                85                  90                  95 Tyr Ile LeuLeu Phe Asp Glu Tyr Asn Glu Lys Lys Ala Ser Ala Gln            100                 105                 110 Lys Asp Ile LeuIle Arg Ile Leu Asp Asp Gly Val Asn Lys Leu Asn        115                 120                 125 Glu Ala Gln Lys SerLeu Leu Gly Ser Ser Gln Ser Phe Asn Asn Ala    130                 135                 140 Ser Gly Lys Leu Leu AlaLeu Asp Ser Gln Leu Thr Asn Asp Phe Ser145                 150                 155                 160 Glu LysSer Ser Tyr Phe Gln Ser Gln Val Asp Arg Ile Arg Lys Glu                165                 170                 175 Ala Tyr AlaGly Ala Ala Ala Gly Ile Val Ala Gly Pro Phe Gly Leu            180                 185                 190 Ile Ile Ser TyrSer Ile Ala Ala Gly Val Ile Glu Gly Lys Leu Ile        195                 200                 205 Pro Glu Leu Asn AspArg Leu Lys Ala Val Gln Asn Phe Phe Thr Ser    210                 215                 220 Leu Ser Val Thr Val LysGln Ala Asn Lys Asp Ile Asp Ala Ala Lys225                 230                 235                 240 Leu LysLeu Ala Thr Glu Ile Ala Ala Ile Gly Glu Ile Lys Thr Glu                245                 250                 255 Thr Glu ThrThr Arg Phe Tyr Val Asp Tyr Asp Asp Leu Met Leu Ser            260                 265                 270 Leu Leu Lys GlyAla Ala Lys Lys Met Ile Asn Thr Cys Asn Glu Tyr        275                 280                 285 Gln Gln Arg His GlyLys Lys Thr Leu Leu Glu Val Pro Asp Ile    290                 295                 300

The S. paratyphi ClyA protein is encoded by the nucleic acid sequence ofSEQ ID NO: 6:

ggaggcaata ggtaggaata agttataaaa caatagctta attgcaattt atatatttaa 60agaggcaaat gattatgact ggaatatttg cagaacaaac tgtagaggta gttaaaagcg 120cgatcgaaac cgcagatggg gcattagatt tttataacaa atacctcgac caggttatcc 180cctggaagac ctttgatgaa accataaaag agttaagccg ttttaaacag gagtactcgc 240aggaagcttc tgttttagtt ggtgatatta aagttttgct tatggacagc caggataagt 300attttgaagc gacacaaact gtttatgaat ggtgtggtgt cgtgacgcaa ttactctcag 360cgtatatttt actatttgat gaatataatg agaaaaaagc atcagcgcag aaagacattc 420tcatcaggat attagatgat ggcgtcaata aactgaatga agcgcaaaaa tctctcctgg 480gaagttcaca aagtttcaac aacgcttcag gaaaactgct ggcattagat agccagttaa 540ctaatgattt ctcggaaaaa agtagttatt tccagtcaca ggtggataga attcgtaagg 600aagcttatgc cggtgctgca gcaggcatag tcgccggtcc gtttggatta attatttcct 660attctattgc tgcgggcgtg attgaaggga aattgattcc agaattgaat gacaggctaa 720aagcagtgca aaatttcttt actagcttat cagtcacagt gaaacaagcg aataaagata 780tcgatgcggc aaaattgaaa ttagccactg aaatagcagc aattggggag ataaaaacgg 840aaaccgaaac aaccagattc tacgttgatt atgatgattt aatgctttct ttactaaaag 900gagctgcaaa gaaaatgatt aacacctgta atgaatacca acaaaggcac ggtaagaaga 960cgcttctcga ggttcctgac atctgataca ttttcattcg ctctgtttac ttttaacgcc 1020cgatagcgtg aagaaaatga gagacggaga aaaagcgata ttcaacagcc cgataaacaa 1080gagtcgttac cgggctggcg ag 1102

In another embodiment of the present invention, the fusion proteincomprises either a full length or polypeptide fragment, analogue orderivative thereof of the ClyA protein Shigella flexneri (GenbankAccession No. AF200955). The amino acid sequence of the S. flexneriaClyA protein is set forth below as SEQ ID NO: 7:

Met Thr Glu Ile Val Ala Asp Lys Thr Val Glu Val Val Lys Asn Ala1               5                   10                  15 Ile Glu ThrAla Asp Gly Ala Leu Asp Leu Tyr Asn Lys Tyr Leu Asp            20                  25                  30 Gln Val Ile ProTrp Gln Thr Phe Asp Glu Thr Ile Lys Glu Leu Ser        35                  40                  45 Arg Phe Lys Gln GluTyr Ser Gln Ala Ala Ser Val Leu Val Gly Asp    50                  55                  60 Ile Lys Thr Leu Leu MetAsp Ser Gln Asp Lys Tyr Phe Glu Ala Thr65                  70                  75                  80 Gln ThrVal Tyr Glu Trp Cys Gly Val Ala Thr Gln Leu Leu Ala Ala                85                  90                  95 Tyr Ile LeuLeu Phe Asp Glu Tyr Asn Glu Lys Lys Ala Ser Ala Pro            100                 105                 110 His

The S. flexneria ClyA protein is encoded by the nucleic acid sequence ofSEQ ID NO: 8:

atgactgaaa tcgttgcaga taaaacggta gaagtagtta aaaacgcaat cgaaaccgca 60gatggagcat tagatcttta taataaatat ctcgatcagg tcatcccctg gcagaccttt 120gatgaaacca taaaagagtt aagtcgcttt aaacaggagt attcacaggc agcctccgtt 180ttagtcggcg atattaaaac cttacttatg gatagccagg ataagtattt tgaagcaacc 240caaacagtgt atgaatggtg tggtgttgcg acgcaattgc tcgcagcgta tattttgcta 300tttgatgagt acaatgagaa gaaagcatcc gcccctcatt aaggtactgg atgacggcat 360cacgaagctg aatgaagcgc aaaattccct gctggtaagc tcacaaagtt tcaacaacgc 420ttccgggaaa ctgctggcgt tagatagcca gttaaccaat gatttttcag aaaaaagcag 480ctatttccag tcacaggtag ataaaatcag gaaggaagcg tatgccggtg ccgcagccgg 540tgtcgtcgcc ggtccatttg gtttaatcat ttcctattct attgctgcgg gcgtagttga 600agggaaactg attccagaat tgaagaacaa gttaaaatct gtgcagagtt tctttaccac 660cctgtctaac acggttaaac aagcgaataa agatatcgat gccgccaaat tgaaattaac 720caccgaaata gccgccatcg gggagataaa aacggaaact gaaaccacca gattctatgt 780tgattatgat gatttaatgc tttctttgct aaaagcagcg gccaaaaaaa tgattaacac 840ctgtaatgag tatcagaaaa gacacggtaa aaagacactc tttgaggtac ctgaagtctg 900ataa 904

In another embodiment, the fusion protein of the present invention,comprising either a full length or polypeptide fragment, analogue orderivative thereof of the ClyA protein is derived from a ClyA consensussequence. A ClyA amino acid consensus sequence derived from thealignment of SEQ ID NOs: 1, 3, 5, and 7 is set forth below as SEQ IDNO:9:

Met Thr Xaa Ile Xaa Ala Xaa Xaa Thr Val Glu Val Val Lys Xaa Ala1               5                   10                  15 Ile Glu ThrAla Asp Gly Ala Leu Asp Xaa Tyr Asn Lys Tyr Leu Asp            20                  25                  30 Gln Val Ile ProTrp Xaa Thr Phe Asp Glu Thr Ile Lys Glu Leu Ser        35                  40                  45 Arg Phe Lys Gln GluTyr Ser Gln Xaa Ala Ser Val Leu Val Gly Asp    50                  55                  60 Ile Lys Xaa Leu Leu MetAsp Ser Gln Asp Lys Tyr Phe Glu Ala Thr65                  70                  75                  80 Gln ThrVal Tyr Glu Trp Cys Gly Val Xaa Thr Gln Leu Leu Xaa Ala                85                  90                  95 Tyr Ile LeuLeu Phe Asp Glu Tyr Asn Glu Lys Lys Ala Ser Ala Xaa            100                 105                 110 Xaa

The ClyA consensus sequence of SEQ ID NO:9 is encoded by the nucleicacid sequence set forth in SEQ ID NO:10 below:

natgacngna atnnttgcag annaaacngt agangtagtt aaaancgcna tcgaaaccgc 60agatggngca ttagatnttt ataanaaata nctcgancag gtnatcccct ggnagacctt 120tgatgaaacc ataaaagagt taagncgntt taaacaggag tantcncagg nagcntcngt 180tttagtnggn gatattaaan nnttncttat gganagccag ganaagtatt ttgaagcnac 240ncaaacngtn tatgaatggt gtggtgtngn gacgcaattn ctcncagcgt atattttnct 300atttgatgan tanaatgaga anaaagcatc ngcncnnnnn nnnnnnctca tnangntant 360ngatganggn ntcannaanc tgaatgaagc gcaaaantcn ctnctgnnaa gntcacaaag 420tttcaacaac gcttcnggna aactgctggc nttagatagc cagttaacna atgatttntc 480ngaaaaaagn agntatttcc agtcacaggt ngatanaatn ngnaaggaag cntatgccgg 540tgcngcagcn ggnntngtcg ccggtccntt tggnttaatn atttcctatt ctattgctgc 600gggcgtnntt gaaggnaaan tgattccaga attgaannac angntaaaan cngtgcanan 660tttctttacn ancntntcnn nnacngtnaa acaagcgaat aaagatatcg atgcngcnaa 720attgaaatta nccacngaaa tagcngcnat nggngagata aaaacggaaa cngaaacnac 780cagattctan gttgattatg atgatttaat gctttctttn ntaaaagnag cngcnaanaa 840aatgattaac acctgtaatg antancanna aagncacggt aanaagacnc tnntngaggt 900ncctganntc tgatan 916

An alternative ClyA amino acid consensus sequence derived from thealignment of SEQ ID NOs: 1, 3, and 5 is set forth below as SEQ ID NO:11:

Met Thr Xaa Ile Xaa Ala Xaa Xaa Thr Val Glu Val Val Lys Xaa Ala1               5                   10                  15 Ile Glu ThrAla Asp Gly Ala Leu Asp Xaa Tyr Asn Lys Tyr Leu Asp            20                  25                  30 Gln Val Ile ProTrp Xaa Thr Phe Asp Glu Thr Ile Lys Glu Leu Ser        35                  40                  45 Arg Phe Lys Gln GluTyr Ser Gln Xaa Ala Ser Val Leu Val Gly Asp    50                  55                  60 Ile Lys Xaa Leu Leu MetAsp Ser Gln Asp Lys Tyr Phe Glu Ala Thr65                  70                  75                  80 Gln ThrVal Tyr Glu Trp Cys Gly Val Xaa Thr Gln Leu Leu Xaa Ala                85                  90                  95 Tyr Ile LeuLeu Phe Asp Glu Tyr Asn Glu Lys Lys Ala Ser Ala Gln            100                 105                 110 Lys Asp Ile LeuIle Xaa Xaa Leu Asp Asp Gly Xaa Xaa Lys Leu Asn        115                 120                 125 Glu Ala Gln Lys SerLeu Leu Xaa Ser Ser Gln Ser Phe Asn Asn Ala    130                 135                 140 Ser Gly Lys Leu Leu AlaLeu Asp Ser Gln Leu Thr Asn Asp Phe Ser145                 150                 155                 160 Glu LysSer Ser Tyr Phe Gln Ser Gln Val Asp Xaa Ile Arg Lys Glu                165                 170                 175 Ala Tyr AlaGly Ala Ala Ala Gly Xaa Val Ala Gly Pro Phe Gly Leu            180                 185                 190 Ile Ile Ser TyrSer Ile Ala Ala Gly Val Xaa Glu Gly Lys Leu Ile        195                 200                 205 Pro Glu Leu Xaa XaaXaa Leu Lys Xaa Val Gln Asn Phe Phe Thr Xaa    210                 215                 220 Leu Ser Xaa Thr Val LysGln Ala Asn Lys Asp Ile Asp Ala Ala Lys225                 230                 235                 240 Leu LysLeu Xaa Thr Glu Ile Ala Ala Ile Gly Glu Ile Lys Thr Glu                245                 250                 255 Thr Glu ThrThr Arg Phe Tyr Val Asp Tyr Asp Asp Leu Met Leu Ser            260                 265                 270 Leu Leu Lys XaaAla Ala Lys Lys Met Ile Asn Thr Cys Asn Glu Tyr        275                 280                 285 Gln Xaa Arg His GlyLys Lys Thr Leu Xaa Glu Val Pro Xaa Xaa    290                 295                 300

The ClyA consensus sequence of SEQ ID NO:11 is encoded by the nucleicacid sequence set forth in SEQ ID NO:12 below:

ngangnaana nntannaata nnttntaaaa cannnnnttn attncaattt atatatttaa 60agaggcnaat gattatgacn gnaatnnttg cagannaaac ngtagangta gttaaaancg 120cnatcgaaac cgcagatggn gcattagatn tttataanaa atanctcgan caggtnatcc 180cctggnagac ctttgatgaa accataaaag agttaagncg ntttaaacag gagtantcnc 240aggnagcntc ngttttagtn ggngatatta aannnttnct tatgganagc cagganaagt 300attttgaagc nacncaaacn gtntatgaat ggtgtggtgt ngngacgcaa ttnctcncag 360cgtatatttt nctatttgat gantanaatg agaanaaagc atcngcncag aaagacattc 420tcatnangnt antngatgan ggnntcanna anctgaatga agcgcaaaaa tcnctnctgn 480naagntcaca aagtttcaac aacgcttcng gnaaactgct ggcnttagat agccagttaa 540cnaatgattt ntcngaaaaa agnagntatt tccagtcaca ggtngatana atnngnaagg 600aagcntatgc cggtgcngca gcnggnntng tcgccggtcc ntttggatta atnatttcct 660attctattgc tgcgggcgtn nttgaaggna aantgattcc agaattgaan nacangntaa 720aancngtgca naatttcttt acnancntnt cnnnnacngt naaacaagcg aataaagata 780tcgatgcngc naaattgaaa ttanccacng aaatagcngc natnggngag ataaaaacgg 840aaacngaaac aaccagattc tacgttgatt atgatgattt aatgctttct ttnntaaaag 900nagcngcnaa naaaatgatt aacacctgta atgantanca nnaaagncac ggtaanaaga 960cnctnntnga ggtncctgan ntctgatann nnntnattcn ntcnntntac tnnnaangnn 1020ngatanngtn nannanatn 1039

In the ClyA amino acid consensus sequences provided supra, the Xaaresidues can be any amino acid, but preferably a neutral or hydrophobicamino acid. In the ClyA nucleic acid consensus sequences, the n residuecan be any nucleic acid.

As discussed supra, the ClyA fusion protein of the present invention maycomprise a full length ClyA protein, analogue or derivative thereof. Inanother embodiment, the fusion protein of the present inventioncomprises a peptide or polypeptide fragment of the ClyA protein.Preferred polypeptide fragments of the ClyA protein are those thatretain the capacity to undergo normal cellular transport and outermembrane vesicle (OMV) assembly. The protein or polypeptide fragments ofClyA may comprise any of the wildtype amino acid sequences provided inSEQ ID NOs: 1, 3, 5, or 7 or the consensus sequences of SEQ ID NOs: 9and 11. Alternatively, the ClyA protein or polypeptide fragment maycontain one or more amino acid substitutions or deletions. In apreferred embodiment, the ClyA protein or polypeptide is a variantcontaining amino acid substitutions or deletions which inactivate itshemolytic activity while maintaining its adhesion/invasion activity.Such amino acid substitutions are readily known in the art and include,for example, the triple mutation V185S-A187S-1193S reported by Wallaceet al., “E. coli hemolysin E (HlyE, ClyA, SheA): X-ray Crystal Structureof the Toxin and Observation of Membrane Pores by Electron Microscopy,”Cell 100:265-76 (2000), which is hereby incorporated by reference in itsentirety, or the deletion of amino acids 183-202 within thetransmembrane domain, as reported by del Castillo et al., “Secretion ofthe Escherichia coli K-12 SheA Hemolysin is Independent of its CytolyticActivity,” FEMS Microbiol. Lett. 204:281-285 (2001), which is herebyincorporated by reference in its entirety.

The ClyA fusion protein used in the accordance with the methods andcompositions of the present invention further comprises at least aportion of a second protein (i.e. a fusion partner). In one embodimentthe second protein is a marker protein. Marker proteins are well know inthe art and include affinity protein markers, such as chitin bindingprotein, maltose binding protein, glutathione-s-transferase, and thepoly(His) tag; epitope markers, such as the V5-tag, c-myc-tag or theHA-tag; and fluorescence protein markers such as the green fluorescentprotein and variants thereof (e.g. blue fluorescent protein, yellowfluorescent protein, and cyan fluorescent protein). Many additionalfluorescence protein markers are well known in the art and commerciallyavailable including, but not limited to, the monomeric Kusabira Orange(mKO) protein, Midori-Ishi cyan fluorescent protein, mCherry redfluorescent protein and the monomeric teal fluorescent protein.

In another embodiment, the second protein of the ClyA fusion proteincomprises a ligand binding protein. Suitable ligand binding proteins,include high-affinity antibody fragments (e.g., Fab, Fab′ and F(ab′)₂),single-chain Fv antibody fragments, nanobodies or nanobody fragments,fluorobodies, or aptamers. Other ligand binding proteins includebiotin-binding proteins, lipid-binding proteins, periplasmic bindingproteins, lectins, serum albumins, enzymes, phosphate and sulfatebinding proteins, immunophilins, metallothionein, or various otherreceptor proteins.

The ClyA fusion protein of the present invention can further comprise atleast a portion of an antigenic protein or peptide. Suitable antigenicproteins or peptides are those derived from pathogenic bacterial, fungalor viral organisms such as Streptococcus species, Candida species,Brucella species, Salmonella species, Shigella species, Pseudomonasspecies, Bordetella species, Clostridium species, Norwalk virus,Bacillus anthracis, Mycobacterium tuberculosis, human immunodeficiencyvirus (HIV), Chlamydia species, human Papillomaviruses, Influenza virus,Paramyxovirus species, Herpes virus, Cytomegalovirus, Varicella-Zostervirus, Epstein-Barr virus, Hepatitis viruses, Plasmodium species,Trichomonas species. Other suitable antigenic proteins or peptidesinclude sexually transmitted disease agents, viral encephalitis agents,protozoan disease agents, fungal disease agents, bacterial diseaseagents, or cancer cell antigens (e.g. prostate specific antigen, TAG-72and CEA, MAGE-1 and tyrosinase), transplant antigens (e.g. CD3 receptor)or autoimmune antigens (e.g. IAS chain), and combinations thereof.

The second protein of the ClyA fusion protein can also comprise atherapeutic protein. A therapeutic protein in the context of the presentinvention is any recombinant protein useful in treating a subjectsuffering from a condition amenable to protein therapy treatment. Suchconditions include, but are in no way limited to, cancer, heart attack,stroke, cystic fibrosis, Gaucher's disease, diabetes, anaemia, andhaemophilia.

A therapeutic protein may be an immunoregulatory molecule. Suitableimmunoregulatory molecules include, but are not limited to, growthfactors, such as M-CSF, GM-CSF; and cytokines, such as IL-2, IL-4, IL-5,IL-6, IL-10, IL-12 or IFN-gamma.

The ClyA fusion proteins used in accordance with the methods of thepresent invention can be generated as described herein or using anyother standard technique known in the art. For example, the fusionpolypeptide can be prepared by translation of an in-frame fusion of thepolynucleotide sequences, i.e., a hybrid gene. The hybrid gene encodingthe fusion polypeptide is inserted into an expression vector which isused to transform or transfect a host cell. Alternatively, thepolynucleotide sequence encoding the ClyA polypeptide or protein isinserted into an expression vector in which the polynucleotide encodingthe second polypeptide is already present. The second polypeptide orprotein of the fusion protein can be fused to the N-, or preferably, tothe C-terminal end of the ClyA polypeptide or protein.

Fusions between the ClyA protein or polypeptide and a second protein orpolypeptide may be such that the amino acid sequence of the ClyA proteinor polypeptide is directly contiguous with the amino acid sequence ofthe second protein. Alternatively, the ClyA portion may be coupled tothe second protein or polypeptide by way of a linker sequence such asthe flexible 5-residue Gly linker described herein or the flexiblelinkers from an immunoglobulin disclosed in U.S. Pat. No. 5,516,637 toHuang et al, which is hereby incorporated by reference in its entirety.The linker may also contain a protease-specific cleavage site so thatthe second protein may be controllably released from ClyA. Examples ofprotease sites include those specific to cleavage by factor Xa,enterokinase, collagenase, Igase (from Neisseria gonorrhoeae),thrombine, and TEV (Tobacco Etch Virus) protease.

Once the fusion protein is constructed, the nucleic acid constructencoding the protein is inserted into an expression system to which themolecule is heterologous. The heterologous nucleic acid molecule isinserted into the expression system or vector in proper sense (5′→3′)orientation relative to the promoter and any other 5′ regulatorymolecules, and correct reading frame. The preparation of the nucleicacid constructs can be carried out using standard cloning methods wellknown in the art as described by Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Springs Laboratory Press, Cold Springs Harbor,N.Y. (1989), which is hereby incorporated by reference in its entirety.U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporatedby reference in its entirety, also describes the production ofexpression systems in the form of recombinant plasmids using restrictionenzyme cleavage and ligation with DNA ligase.

Suitable expression vectors include those which contain replicon andcontrol sequences that are derived from species compatible with the hostcell. For example, if E. coli is used as a host cell, plasmids such aspUC19, pUC18 or pBR322 may be used.

Different genetic signals and processing events control many levels ofgene expression (e.g., DNA transcription and messenger RNA (“mRNA”)translation) and subsequently the amount of fusion protein that isdisplayed on the cell or vesicle surface. Transcription of DNA isdependent upon the presence of a promoter, which is a DNA sequence thatdirects the binding of RNA polymerase, and thereby promotes mRNAsynthesis. Promoters vary in their “strength” (i.e., their ability topromote transcription). For the purposes of expressing a cloned gene, itis desirable to use strong promoters to obtain a high level oftranscription and, hence, expression and surface display. Depending uponthe host system utilized, any one of a number of suitable promoters maybe used. For instance, when using E. coli, its bacteriophages, orplasmids, promoters such as the T7 phage promoter, lac promoter, trppromoter, recA promoter, ribosomal RNA promoter, the P_(R) and P_(L)promoters of coliphage lambda and others, including but not limited, tolacUV 5, ompF, bla, lpp, and the like, may be used to direct high levelsof transcription of adjacent DNA segments. Additionally, a hybridtrp-lacUV5 (tac) promoter or other E. coli promoters produced byrecombinant DNA or other synthetic DNA techniques may be used to providefor transcription of the inserted gene.

Translation of mRNA in prokaryotes depends upon the presence of theproper prokaryotic signals, which differ from those of eukaryotes.Efficient translation of mRNA in prokaryotes requires a ribosome bindingsite called the Shine-Dalgarno (“SD”) sequence on the mRNA. Thissequence is a short nucleotide sequence of mRNA that is located beforethe start codon, usually AUG, which encodes the amino-terminalmethionine of the protein. The SD sequences are complementary to the3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding ofmRNA to ribosomes by duplexing with the rRNA to allow correctpositioning of the ribosome. For a review on maximizing gene expression,see Roberts and Lauer, Methods in Enzymology, 68:473 (1979), which ishereby incorporated by reference in its entirety.

Host cells suitable for expressing and displaying the ClyA fusion on thecell surface or cell vesicle surface include any one of the morecommonly available gram negative bacteria. Suitable microorganismsinclude Pseudomonas aeruginosa, Escherichia coli, Salmonellagastroenteritis (typhimirium), S. typhi, S. enteriditis, Shigellaflexneri, S. sonnie, S dysenteriae, Neisseria gonorrhoeae, N.meningitides, Haemophilus influenzae H. pleuropneumoniae, Pasteurellahaemolytica, P. multilocida, Legionella pneumophila, Treponema pallidum,T. denticola, T. orates, Borrelia burgdorferi, Borrelia spp. Leptospirainterrogans, Klebsiella pneumoniae, Proteus vulgaris, P. morganii, P.mirabilis, Rickettsia prowazeki, R. typhi, R. richettsii, Porphyromonas(Bacteriodes) gingivalis, Chlamydia psittaci, C. pneumoniae, C.trachomatis, Campylobacter jejuni, C. intermedis, C. fetus, Helicobacterpylori, Francisella tularenisis, Vibrio cholerae, Vibrioparahaemolyticus, Bordetella pertussis, Burkholderie pseudomallei,Brucella abortus, B. susi, B. melitens is, B. can is, Spirillum minus,Pseudomonas mallei, Aeromonas hydrophila, A salmonicida, and Yersiniapestis. Methods for transforming/transfecting host cells with expressionvectors are well-known in the art and depend on the host system selectedas described in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Springs Laboratory Press, Cold Springs Harbor, N.Y. (1989), whichis hereby incorporated by reference in its entirety.

Following transformation of the host cell with an expression vectorcomprising the nucleic acid construct encoding the ClyA fusion protein,the CylA fusion protein is expressed and displayed on the cell surfaceas well as the surface of outer membrane vesicles.

In one embodiment of the present invention, a plurality of proteins orpolypeptides are displayed on the surface of a plurality of cells orcell vesicles. The plurality of proteins or polypeptides displayed onthe cell or cell vesicle surface are ClyA fusion proteins where eachClyA fusion protein has a different second protein. The plurality ofClyA fusion proteins forms a library of proteins or peptides that areamenable to cell or cell vesicle surface display.

The cell and vesicle surface display of polypeptide or protein librariesgenerated in accordance with the methods of the present invention can beused to facilitate the identification of high affinity antibodies,antibody targets, or other specific ligand binding proteins or smallmolecules. In addition to facilitating the identification ofprotein-ligand binding interactions, cell and cell vesicle surfacedisplay of polypeptides can also be used to assay for other desirableprotein properties including catalytic activity, inhibitory activity,and altered structural conformations.

In a preferred embodiment of the present invention, the library ofpolypeptides displayed on the cell or vesicle surface comprise a libraryof antibodies, antibody fragments, or fluorobodies. As shown herein, thefusion of ClyA to a nucleic acid encoding an antibody facilitatesantibody display on the surface of a host cell or cellular vesicle.Nucleic acids encoding antibodies or antibody fragments can be obtainedfrom an animal immunized with a selected antigen; alternatively,antibody genes from other sources can be used, such as those produced byhybridomas or produced by mutagenesis of a known antibody gene. Onepreferred method of obtaining nucleic acid segments is to isolate mRNAfrom antibody cells of an immunized animal. The mRNA may be amplified,for example by PCR, and used to prepare DNA segments to be used asfusion partners to ClyA. DNA segments that have been mutagenized fromone or more DNAs that encode a selected antibody or antibody fragmentmay also be used.

Once an antibody expression library is prepared, the selected antigenfor which one desires to identify and isolate specific antibody orantibodies is labeled with a detectable label. There are many types ofdetectable labels, including fluorescent labels (e.g fluoresceinisothiocyanate (FITC), Alexa Fluor 488, phycoerytherin (PE), PE-TexasRed, PE-Cy5, PerCP, PerCP-Cy5.5, and PE-Cy7). The labeled antigen iscontacted with the cells displaying the antibody expression libraryunder conditions that allow specific antigen-antibody binding.Conditions can be varied so that only very tightly binding interactionsoccur; for example, by using very low concentrations of labeled antigen.

Identifying the antibody or antibody fragment expressing cells may beaccomplished by methods that depend on detecting the presence of thebound detectable label. A particularly preferred method foridentification and isolation is cell sorting or flow cytometry such asFluorescent Activated Cell Sorting (FACS).

The present invention is also directed to a method of screening alibrary of candidate compounds to identify a compound that binds to atarget protein. This method involves providing the library of candidatecompounds to be screened and a cell or a cell vesicle displaying a ClyAfusion protein. The ClyA fusion protein comprises at least a portion ofthe ClyA protein and at least a portion of a second protein, where thesecond protein of the ClyA fusion protein comprises the target protein.The method further includes contacting the library of candidatecompounds with the cell or cell vesicle displaying the ClyA fusiontarget protein under conditions effective for the candidate compound tobind to the target protein and identifying those compounds that bind tothe target protein.

Another aspect of the present invention relates to methods of imagingcells. This method involves providing either a fusion protein containingat least a portion of the ClyA protein and a marker protein coupled tosaid ClyA protein or a nucleic acid construct encoding the fusionprotein. The method further involves administering to a cell the fusionprotein or the nucleic acid construct under conditions effective todisplay the fusion protein on the cell and imaging the cell based on thepresence of the marker protein.

Any of the marker proteins described supra can be used as the ClyAfusion partner to facilitate the method of imaging cells. In a preferredembodiment, the marker protein is a fluorescent marker protein. Asdiscussed supra, there are a number of fluorescence protein markers thatare well known in the art and commercially available, including forexample, Green Fluorescent Protein (GFP) and all of its variants (e.g.BlueFP, YellowFP, CyanFP) that would facilitate the imaging of cells inaccordance with this aspect of the invention.

Cell imaging can be achieved using any fluorescence based microscopymethod known in the art including, but not limited to, epifluorescencemicroscopy, two-photon excitation microscopy, or confocal microscopy.

Another aspect of the present invention relates to a method of sortingcells. This method involves providing either a fusion protein containingat least a portion of the ClyA protein and a marker protein coupled tosaid ClyA protein or a nucleic acid construct encoding the fusionprotein. The method further involves administering to a cell the fusionprotein or the nucleic acid construct under conditions effective todisplay the fusion protein on the cell and sorting the cell based on thepresence of the marker protein.

Any of the marker proteins described supra can be used as the ClyAfusion partner to facilitate the method of sorting cells. For example,the second protein of the fusion protein can comprise a marker proteinhaving a polyhistidine-tag (His-tag). Cells displaying the fusionprotein and, therefore, the His-tag can be readily sorted using affinitypurification media such as, NTA-agarose, HisPur resin or Talon resin.Like the his-tag, other protein marker “tags” including the V5-tag,c-myc-tag or the HA-tag can also be adapted for cell sorting purposes.

The marker protein in accordance with this aspect of the invention canalso be any ligand or ligand binding protein which can be sorted basedon its selective binding to its respective binding partner. Magneticactivated cell sorting (MACS) using Dynal® Dynabeads® is an exemplarymethod of cell sorting based on this methodology. Dynabeads® are smallmagnetic beads which are covered with any desired ligand (e.g. antibody,protein, or antigen) having affinity for the target marker protein. Oncethe target protein is bound to the Dynabead®, the beads are bound to amagnetic column, which removes the target protein (and cell) from amixed sample or solution. The bead bound cells are then eluted from themagnetic column.

In a preferred embodiment, the marker protein is a fluorescent markerprotein and the cells are sorted by FACS. Any of the fluorescenceprotein markers discussed supra are suitable for facilitating thesorting of cells in accordance with this aspect of the invention.Generally, fluorescent proteins are chosen which have an excitationwavelength that is matched to the wavelength of illuminating light(typically in the range of about 485 to about 491 nm) and emissionspectra that can be detected by an appropriate detector device. By wayof example, many fluorescent proteins have an emission maxima in a rangeof about 510 to about 750 nm.

The optical detection systems used for cell sorting have one or morelight sources, preferably in the form of one or more amplified orcollimated beams of light, that are able to excite the fluorescentmarker protein; and one or more detectors that are able to detect thefluorescence emissions caused by the marker protein. Suitable opticaldetection systems include, without limitation, single-laser flowcytometers; dual- or multiple-laser flow cytometers; and hematologyanalyzers equipped with an appropriate illumination device (e.g., diode,laser, etc.).

Another aspect of the present invention relates to a method ofdelivering a therapeutic agent to a cell, which involves providing avesicle displaying a ClyA fusion protein, where the ClyA fusion proteincomprises at least a portion of the ClyA protein and at least a portionof a second protein. The vesicle contains the therapeutic agent to bedelivered and the second protein of the ClyA fusion protein comprises atargeting protein. The vesicle is administered to a cell underconditions effective to deliver the therapeutic agent to the cell.

Therapeutic agents may be encapsulated in membrane vesicles by culturingthe microorganisms capable of producing membrane vesicles in thepresence of the therapeutic agents. Membrane vesicles are typicallyobtained from gram-negative bacteria. Suitable microorganisms forproducing the membrane vesicles include, but not limited to, Pseudomonasaeruginosa, Escherichia coli, Salmonella gastroenteritis (typhimirium),S. typhi, S. enteriditis, Shigella flexneri, S. sonnie, S dysenteriae,Neisseria gonorrhoeae, N. meningitides, Haemophilus influenzae H.pleuropneumoniae, Pasteurella haemolytica, P. multilocida, Legionellapneumophila, Treponema pallidum, T. denticola, T. orales, Borreliaburgdorferi, Borrelia spp. Leptospira interrogans, Klebsiellapneumoniae, Proteus vulgaris, P. morganii, P. mirabilis, Rickettsiaprowazeki, R. typhi, R. richettsii, Porphyromonas (Bacteriodes)gingivalis, Chlamydia psittaci, C. pneumoniae, C. trachomatis,Campylobacter jejuni, C. intermedis, C. fetus, Helicobacter pylori,Francisella tularenisis, Vibrio cholerae, Vibrio parahaemolyticus,Bordetella pertussis, Burkholderie pseudomallei, Brucella abortus, B.susi, B. melitens is, B. can is, Spirillum minus, Pseudomonas mallei,Aeromonas hydrophila, A salmonicida, and Yersinia pestis. Therapeuticagents may also be produced by the microorganism by transforming themicroorganism with a gene which expresses the therapeutic agentpreferably in the periplasmic space.

Any of a wide variety of therapeutic agents may be encapsulated incellular vesicles including antimicrobial agents, metabolic regulators,immune modulators, antiproliferative agents, chemotherapeutics, etc. Thetherapeutic agent can be a nucleic acid molecule, protein, or smallmolecule.

The ClyA fusion protein displayed on the cell vesicle surface willtarget the therapeutic agent to the tissue where it is most needed. Thesecond protein of the ClyA fusion protein comprises the target protein.Suitable target proteins include any of the ligand binding proteinsdescribed supra, especially antibodies or antibody fragments directed tocell specific surface receptors and proteins. Alternatively, the targetprotein can be any ligand that will bind to a cell-specific surfacereceptor. Targeting the vesicle containing the therapeutic agent to onlythe tissues at risk reduces the exposure of other tissues to potentialtoxic side effects of the therapeutic agent. Slow sustained release oftherapeutic agents from vesicles will also prolong the residence time ofthe therapeutic agent in areas where it is most needed.

The present invention is also directed to a method of eliciting animmune response in a mammal. This method involves providing a cell or acell vesicle displaying a ClyA fusion protein. The ClyA fusion proteincomprises at least a portion of the ClyA protein and at least a portionof a second protein, where the second protein of the ClyA fusion proteincomprises an antigenic protein or peptide capable of eliciting an immuneresponse in the mammal. The cell or vesicle is administered to themammal under conditions effective to elicit the immune response.

Any antigenic protein or peptide capable of eliciting an immune responsein a mammal can be used in accordance with this aspect of the presentinvention. A number of exemplary antigens derived from infectious orpathogenic bacterial, fungal or viral organisms, as well as, tumor cellspecific, autoimmune or transplant antigens are described supra.

The present invention is also directed to drug and vaccine deliveryvehicles consisting of a cell vesicles displaying a ClyA fusion protein.The ClyA fusion protein comprises at least a portion of the ClyA proteinand at least a portion of a second protein.

Drug delivery vehicles of the present invention comprise the drug ortherapeutic agent to be delivered encapsulated by the cell vesicle asdescribed supra. Suitable drugs or therapeutic agents to be deliveredinclude nucleic acid molecules, e.g. RNAi, therapeutic proteins, orsmall molecules. Delivery of the drug or therapeutic agent to its targetcell is facilitated by display of the ClyA fusion protein on the surfaceof the cell vesicle where the second protein of the ClyA fusion proteincomprises a cell specific targeting protein as described supra.

Vaccine delivery vehicles of the present invention may either beprophylactic (i.e. to prevent infection) or therapeutic (i.e. to treatinfection), but will typically be prophylactic. In this embodiment ofthe present invention, the ClyA fusion protein contains a secondimmunogenic protein or antigen. Suitable antigenic proteins and peptidesare described supra. In a preferred embodiment, the second protein ofthe ClyA fusion protein is a vaccine subunit protein. Immunogeniccompositions used as vaccines comprise an immunologically effectiveamount of antigen(s), as well as any other components, as needed. Animmunologically effective amount, is the amount administrated to anindividual, either in a single dose or as part of a series, that iseffective for treatment or prevention. The amount varies depending uponthe health and physical condition of the individual to be treated, age,the taxonomic group of individual to be treated (e.g. non-human primate,primate, etc.), the capacity of the individual's immune system tosynthesize antibodies, the degree of protection desired, the formulationof the vaccine, the treating doctor's assessment of the medicalsituation, and other relevant factors. It is expected that the amountwill fall in a relatively broad range that can be determined throughroutine trials.

Methods for preparing cellular vesicles suitable for administration asdrug and vaccine delivery vehicles and methods and formulations foradministration of cellular vesicles are known in the art and describedherein and in WO2002/0028215 to Kadurugamuwa and Beveridge,WO2006/024946 to Oster et al., and WO2003/051379 to Foster et al., whichare hereby incorporated by reference in their entirety.

The drug or vaccine delivery vehicles of the present invention can beformulated into pharmaceutically acceptable compositions for patientadministration. An effective quantity of the active vesicles arecombined with a pharmaceutically acceptable vehicle as described, forexample, in Remington's Pharmaceutical Sciences (Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985,which is hereby incorporated by reference in its entirety). On thisbasis, the pharmaceutical compositions include, albeit not exclusively,solutions of the membrane vesicles in association with one or morepharmaceutically acceptable vehicles or diluents, and contained inbuffered solutions with a suitable pH and isoosmotic with thephysiological fluids.

The vesicle delivery vehicles of the present invention can beadministered orally, parenterally, for example, subcutaneously,intravenously, intramuscularly, intraperitoneally, by intranasalinstillation, or by application to mucous membranes, such as, that ofthe nose, throat, and bronchial tubes. They may be administered alone orwith suitable pharmaceutical carriers, and can be in solid or liquidform such as, tablets, capsules, powders, solutions, suspensions, oremulsions.

The delivery vehicles of the present invention may be orallyadministered, for example, with an inert diluent, or with an assimilableedible carrier, or they may be enclosed in hard or soft shell capsules,or they may be compressed into tablets, or they may be incorporateddirectly with the food of the diet. For oral therapeutic administration,the delivery vehicles may be incorporated with excipients and used inthe form of tablets, capsules, elixirs, suspensions, syrups, and thelike. Such compositions and preparations should contain at least 0.1% ofdelivery vehicle. The percentage of the delivery vehicle carrying thedrug or vaccine in these compositions may, of course, be varied and mayconveniently be between about 2% to about 60% of the weight of the unit.The amount of drug or vaccine in such therapeutically usefulcompositions is such that a suitable dosage will be obtained. Preferredcompositions according to the present invention are prepared so that anoral dosage unit contains between about 1 and 250 mg of active drug orvaccine.

The tablets, capsules, and the like may also contain a binder such asgum tragacanth, acacia, corn starch, or gelatin; excipients such asdicalcium phosphate; a disintegrating agent such as corn starch, potatostarch, alginic acid; a lubricant such as magnesium stearate; and asweetening agent such as sucrose, lactose, or saccharin. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier, such as a fatty oil.

Various other materials may be present as coatings or to modify thephysical form of the dosage unit. For instance, tablets may be coatedwith shellac, sugar, or both. A syrup may contain, in addition to activeingredient, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye, and flavoring such as cherry or orange flavor.

The delivery vehicles containing a therapeutic agent or carrying avaccine antigen may also be administered parenterally. Solutions orsuspensions of these vehicles can be prepared in water suitably mixedwith a surfactant, such as hydroxypropylcellulose. Dispersions can alsobe prepared in glycerol, liquid polyethylene glycols, and mixturesthereof in oils. Illustrative oils are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil, ormineral oil. In general, water, saline, aqueous dextrose and relatedsugar solution, and glycols such as, propylene glycol or polyethyleneglycol, are preferred liquid carriers, particularly for injectablesolutions. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

The delivery vehicles of the present invention may also be administereddirectly to the airways in the form of an aerosol. For use as aerosols,the compounds of the present invention in solution or suspension may bepackaged in a pressurized aerosol container together with suitablepropellants, for example, hydrocarbon propellants like propane, butane,or isobutane with conventional adjuvants. The materials of the presentinvention also may be administered in a non-pressurized form such as ina nebulizer or atomizer.

The following examples illustrate various methods for compositions inthe treatment method of the invention. The examples are intended toillustrate, but in no way limit, the scope of the invention.

EXAMPLES Example 1 Bacterial Strains, Plasmids and Growth Conditions

The bacterial strains and plasmids used in these examples are describedin Table 1.

TABLE 1 Bacterial strains and plasmids Bacterial strain or Source (whichare hereby incorporated by plasmid Genotype/Description reference intheir entirety) 1292 supE hsdS met gal lacY tonA Bernadac et al.,“Escherichia coli tol-pal Mutants Form Outer Membrane Vesicles,” JBacteriol 180: 4872-8 (1998) JC8031 1292 ΔtolRA Bernadac et al.,“Escherichia coli tol-pal Mutants Form Outer Membrane Vesicles,” JBacteriol 180: 4872-8 (1998) BW25113 lacI^(q) rrnB_(T14) ΔlacZ_(WJ16)hsdR514 Datsenko et al., “One-Step Inactivation of ΔaraBAD_(AH33)ΔrhaBAD_(LD78) Chromosomal Genes in Escherichia coli K-12 using PCRProducts,” Proc Natl Acad Sci USA 97: 6640-5 (2000) BW25113 BW25113nlpI::Kan created via Baba et al., “Construction of EscherichianlpI::Kan the method of Datsenko and coli K-12 In-Frame, Single-GeneWanner Knockout Mutants: the Keio Collection,” Mol Syst Biol 2: 20060008 (2006) DHB4 MC1000 phoR Δ (phoA) PvuII Δ Laboratory stock (malF)3F′[lacI^(q)ZYA pro] DHA DHB4 dsbA::Kan Qi et al., “DnaK Promotes theSelective Export of Outer Membrane Protein Precursors in SecA-DeficientEscherichia coli,” J Biol Chem 277: 51077-83 (2002) JCA JC8031 dsbA::KanThis study pBAD18-Cm araBAD promoter; pBR322 ori Guzman et al., “TightRegulation, Cm^(r) Modulation, and High-Level Expression by VectorsContaining the Arabinose PBAD Promoter,” J Bacteriol 177: 4121- 30(1995) pBAD18-Kan araBAD promoter; pBR322 ori Guzman et al., “TightRegulation, Cm^(r) Modulation, and High-Level Expression by VectorsContaining the Arabinose PBAD Promoter,” J Bacteriol 177: 4121- 30(1995) pBAD24 araBAD promoter; pBR322 ori Guzman et al., “TightRegulation, Amp^(r) Modulation, and High-Level Expression by VectorsContaining the Arabinose PBAD Promoter,” J Bacteriol 177: 4121- 30(1995) pClyA-His6 E. coli clyA carrying a C-terminal This study 6xpolyhistidine tag cloned in pBAD18-Cm pGFP gfp-mut2 gene cloned inpBAD18- This study Cm pClyA-GFP clyA gene fused to 5′ end of gfp- Thisstudy mut2 in pBAD18-Cm pGFP-ClyA clyA gene fused to 3′ end of gfp- Thisstudy mut2 in pBAD18-Cm pΔss-Bla Mature region of bla gene cloned inpBAD18-Kan pClyA-Bla clyA gene fused to 5′ end of bla in This studypBAD18-Kan pBla-ClyA clyA gene fused to 3′ end of bla in This studypBAD18-Kan pKEG01 Flavobacterium sp. opd gene in Shimazu et al.,“Thermally Triggered pET78UF Purification and Immobilization ofElastin-OPH Fusions,” Biotechnol Bioeng 81: 74-9 (2003) pOPH opd genecloned in pBAD24 This study pClyA-OPH clyA gene fused to 5′ end of opdin This study pBAD24 pOPH-ClyA clyA gene fused to 3′ end of opd in Thisstudy pBAD24 pLacZ lacZ gene cloned in pBAD24 This study pClyA-LacZ clyAgene fused to 5′ end of lacZ This study in pBAD24 pB18D anti-digoxinscFv fused to the 3′ Daugherty et al., “Development of an end oflpp-ompA cloned in Optimized Expression System for the pBAD18 Screeningof Antibody Libraries Displayed on the Escherichia coli Surface,”Protein Eng 12: 613-21 (1999) pB24D anti-digoxin scFv fused to the 3′This study end of lpp-ompA cloned in pBAD24 pscFv.Dig anti-digoxin scFvcloned in This study pBAD24 pClyA-scFv.Dig clyA gene fused to 5′ end ofanti- This study digoxin scFv in pBAD24 pscFv.Dig-ClyA clyA gene fusedto 3′ end of anti- This study digoxin scFv in pBAD24 pClyA(Δ293-303)-clyA with 10 C-terminal residues This study scFv.Dig removedpClyA(Δ156-303)- clyA with 147 C-terminal residues This study scFv.Digremoved pClyA(Y288G)- clyA with Tyr288 residue mutated This studyscFv.Dig to Gly

Strain JCA was made by introducing the dsbA::Kan allele into JC8031cells by P1 vir transduction using DHA as the donor. Plasmid pClyA wasconstructed by ligating the PCR-amplified clyA gene into pBAD18-Cmbetween Sad and XbaI sites. Insertion of DNA encoding either the gfpmut2gene (Crameri et al., “Improved Green Fluorescent Protein by MolecularEvolution Using DNA Shuffling,” Nat Biotechnol 14:315-9 (1996) andDeLisa et al., “Genetic Analysis of the Twin Arginine TranslocatorSecretion Pathway in Bacteria,” J Biol Chem 277:29825-31 (2002), whichare hereby incorporated by reference in their entirety) or a 6×polyhistidine sequence between XbaI and HindIII sites resulted inplasmids pClyA-GFP and pClyA-His6, respectively. Plasmid pGFP-ClyA wasconstructed by first cloning the gfpmut2 gene between Sad and Xmal sitesof pBAD 18-Cm followed by insertion of the clyA gene between XmaI andXbaI sites. Plasmid pGFP was constructed by ligating the PCR-amplifiedgfpmut2 gene between Sad and HindIII sites of pBAD18-Cm. For ClyA-Xfusions in pBAD24, each of the PCR-amplified partner genes (except forbla) was inserted between XmaI and SphI sites followed by clyA ligationbetween NcoI and XmaI sites. X-ClyA fusions were similarly constructedin pBAD24 with the ligations of fusion partner between NcoI and XmaI,and clyA between XmaI and SphI. Control plasmids without clyA wereconstructed by inserting the fusion partner, X, between NcoI and SphI ofpBAD24. For Bla fusions with ClyA, a similar strategy as described abovewas used for inserting clyA and bla into plasmid pBAD18-Kan between Sadand XmaI and SphI sites. The gene encoding the Lpp-OmpA-scFv.Dig chimerain pB18D was amplified and ligated into pBAD24 between NcoI and SphI,resulting in pB24D. To generate pClyA(Δ156-303)-scFv.Dig, pClyA-scFv.Digwas digested with HpaI and XmaI and then self-ligated via blunt-endligation after removal of overhanging basepairs. To generatepClyA(Δ293-303)-scFv.Dig, DNA encoding the first 292 amino acids of ClyAwas PCR-amplified and inserted in place of wt clyA in pClyA-scFv.Dig.Plasmid pClyA(Y288G)-scFv.Dig was generated with pClyA-scFv.Dig astemplate for site-directed mutagenesis using a Stratagene QuickChange®site-directed mutagenesis kit. Cells were grown in LB broth withappropriate antibiotics: ampicillin, 100 ng/ml; chloramphenicol, 25ng/ml; and kanamycin, 50 μg/ml. Cell growth was maintained at 37° C.unless otherwise noted. Protein synthesis was induced for 6 h by adding0.2% arabinose when cells reached an OD₆₀₀≈0.5.

Example 2 Cell Culture

Human epithelial cervical carcinoma (HeLa) cells were obtained from theAmerican Type Culture Collection (ATCC # CCL-2) and grown in Dulbecco'smodified Eagle's minimal essential medium (DMEM) supplemented with 10%NuSerum, and 1% penicillin/streptomycin. Cells were maintained at 37° C.in a humidified atmosphere of 95% air, 5% CO₂. For fluorescencemicroscopy experiments, cells were grown on 12-mm circular glasscoverslips for two days prior to experimentation.

Example 3 Subcellular Fractionation

Cytoplasmic and periplasmic fractions from cells expressing fusionproteins were generated by the cold osmotic shock procedure (Kim et al.,“Twin-Arginine Translocation of Active Human Tissue PlasminogenActivator in Escherichia coli,” Applied and Environmental Microbiology71:8451-8459 (2005)), which is hereby incorporated by reference in itsentirety) and the pellet remaining after removal of the soluble fractionwas collected as the insoluble fraction.

Example 4 Isolation of Bacterial Vesicles

Vesicles were isolated from late log-phase bacterial cultures grownaerobically at 37° C. in LB broth (unless otherwise indicated)essentially as described previously (Wai et al., “Vesicle-MediatedExport and Assembly of Pore-Forming Oligomers of theEnterobacterial ClyACytotoxin,” Cell 115:25-35 (2003), which is hereby incorporated byreference in its entirety). Briefly, bacterial cells were removed bycentrifugation at 5,000×g for 15 min at 4° C. and the cell-freesupernatants were filtered through a 0.2 μm-pore-size vacuum filter.Vesicles were collected from the filtered supernatant byultracentrifugation at 141,000×g for 2 h at 4° C. in a 28 Ti rotor(Beckman Instruments, Inc., Fullerton, Calif.) and the pellet containingOMVs was carefully removed and suspended in PBS (pH 7.0). Vesiclepreparations were plated on LB agar to confirm complete removal ofbacterial cells. Vesicles preparations were kept at −20° C.

Example 5 Outer Membrane Vesicles Fractionation

Separation of pelleted outer membrane vesicle samples was performed asdescribed (Horstman et al., “Enterotoxigenic Escherichia coli SecretesActive Heat-Labile Enterotoxin Via Outer Membrane Vesicles,” J Biol Chem275:12489-96 (2000), which is hereby incorporated by reference in itsentirety). Briefly, vesicles were isolated as described above butsuspended in 50 mM HEPES (pH 6.8), adjusted to 45% Optiprep (Sigma, St.Louis, Mo.) in 0.15 ml and transferred to the bottom of 12-mlultracentrifugation tubes. Different Optiprep/HEPES layers weresequentially added as follows: 0.9 ml 35%, 0.9 ml 30%, 0.66 ml 25%, 0.66ml 20%, 0.33 ml 15% and 0.33 ml 10%. Gradients were centrifuged(180,000×g, 180 min, 4° C.). A total of 10 fractions of equal volumeswere sequentially removed and analysed by SDS-PAGE.

Example 6 Vesicle Characterization

The amount of vesicles in purified cell-free supernatant was determinedby measuring the total protein concentration or the dry mass of vesiclesaccording to published protocols (Kadurugamuwa et al., “VirulenceFactors Are Released from Pseudomonas aeruginosa in Association withMembrane Vesicles During Normal Growth and Exposure to Gentamicin: ANovel Mechanism of Enzyme Secretion,” J Bacteriol 177:3998-4008 (1995),which is hereby incorporated by reference in its entirety). Particlesize distribution and zeta potential of vesicle samples containingapproximately 30 μg/mL total protein in 1 mL of PBS were measured in aNanosizer Nano ZS instrument (Malvern Instruments, Westborough, Ma.)using standard protocols. Malvern Dispersion Technology Software wasused for data acquisition and analysis, applying the general purposealgorithm for calculating size distributions and the Smoluchowskiapproximation for determining zeta potential.

Example 7 Protein Assays

Whole cells, OMVs and subcellular fractions were assayed for Bla, LacZand OPH activity using nitrocefin (Sigma), ONPG (Sigma) and paraoxon(Sigma), respectively, according to standard spectrophotometric assays(Cho et al., “Bacterial Cell Surface Display of OrganophosphorusHydrolase for Selective Screening of Improved Hydrolysis ofOrganophosphate Nerve Agents,” Appl Environ Microbiol 68:2026-30 (2002);Francisco et al., “Transport and Anchoring of Beta-lactamase to theExternal Surface of Escherichia coli,” Proc Natl Acad Sci USA 89:2713-7(1992); and Miller J. H., A Short Course in Bacterial Genetics. ALaboratory Manual and Handbook for Escherichia coli and RelatedBacteria, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1992), which are hereby incorporated by reference in their entirety).Cells expressing anti-digoxin scFv were labeled with Dig-BODIPY andanalyzed by flow cytometry as described (Daugherty et al., “Developmentof an Optimized Expression System for the Screening of AntibodyLibraries Displayed on the Escherichia coli Surface,” Protein Eng12:613-21 (1999), which is hereby incorporated by reference in itsentirety). Total protein concentration was assayed using the BCA ProteinAssay kit (Pierce, Rockford, Ill.). Protease accessibility assays wereperformed as described but with Proteinase K (Kesty et al.,“Incorporation of Heterologous Outer Membrane and Periplasmic Proteinsinto Escherichia coli Outer Membrane Vesicles,” J Biol Chem 279:2069-76(2004), which is hereby incorporated by reference in its entirety).Briefly, vesicles were treated at 37° C. for 30 min in 20 mM Tris HCl(pH 8.0) with PK (0.1 mg/ml) in either the absence or presence of 1%SDS. In parallel control experiments, ClyA-GFP and GFP-ClyA that hadbeen purified by IMAC were similarly treated with PK. Following theincubation, all samples were placed on ice and 1 mM PMSF was added toquench all proteolysis, and the samples were analysed by SDS-PAGE.Western blotting was performed as described by Chen et al., “Isolationof High-Affinity Ligand-Binding Proteins by Periplasmic Expression withCytometric Screening (PECS),” Nat Biotechnol 19:537-42 (2001), which ishereby incorporated by reference in its entirety, using the followingprimary antibodies: anti-ClyA (kindly provided by Sun Nyunt Wai, UmeåUniversity, Sweden), anti-GFP (Sigma), anti-GroEL (Sigma),anti-polyhistidine (Sigma), anti-OmpA and anti-DsbA (kindly provided byJon Beckwith, Harvard Medical School). Membranes were developed on filmusing Immuno-Star™ HRP Substrate Kit (Bio-Rad, Hercules, Calif.).

Example 8 Surface Plasmon Resonance (SPR)

The SPR configuration consisted of a sensor chip, an optical measuringunit, a flow cell and a syringe pump and was similar to that developedpreviously (Baac et al., “Antibody-Based Surface Plasmon ResonanceDetection of Intact Viral Pathogen,” Biotechnol Bioeng 94:815-9 (2006)and Ferracci et al., “Synaptic Vesicle Chips to Assay BotulinumNeurotoxins,” Biochem J 391:659-66 (2005), which are hereby incorporatedby reference in their entirety). The SPR chip was SF10 glass with a thinlayer (50 nm) of gold that was attached to a prism made of SF10 usingindex matching oil. Two microfluidic channels (reference and test) madeof polydimethylsiloxane (PDMS) were placed on the SPR sensor chip andscrew-clamped to seal the channels. Single-wavelength light was obtainedby passing white light from a Xe-lamp (Oriel, Irvine, Calif.) through amonochromater (Oriel). Light with a bandwidth less than 1 nm passedthrough the polarizer, where only p-polarized light was transmitted. Thereflected light intensity (RI) was measured with a CCD camera (Sony)that showed high sensitivity around 600 nm. When the incident angle ofthe beam was fixed to 60 degrees, treatment of the sensor chip with PBSresulted in an SPR wavelength of about 600 nm. The SPR wavelength wasobtained at each pixel by fitting the RI versus wavelength data to asecond-order polynomial equation and the resulting SPR wavelengths,covering a pre-defined region of interest, were averaged. Before eachmeasurement, the RI of s-polarized light was recorded at each pixel forreference. The reliable detection limit of the SPR sensor was measuredto be less than 0.2 nm.

Example 9 Vesicle and Vesicle Antigen Detection Using SPR

An SPR chip for detection of vesicles and vesicle-associated antigenswas performed as follows. First, an alkanethiol monolayer wasself-assembled on the 50-nm gold layer of the sensor chip surface with amixed solution (1:2 molar ratio) of 10 mM 11-mercaptoundecanoic acid(11-MUA) and 6-mercapto-1-hexanol (6-MCH) as described previously (Choiet al., “Enhanced Performance of a Surface Plasmon ResonanceImmunosensor for Detecting Ab-GAD Antibody Based on the ModifiedSelf-Assembled Monolayers,” Biosens Bioelectron 21:378-83 (2005) and Leeet al., “Characterization of a Self-Assembled Monolayer of Thiol on aGold Surface and the Fabrication of a Biosensor Chip Based on SurfacePlasmon Resonance for Detecting anti-GAD Antibody,” Biosens Bioelectron20:1422-7 (2005), which are hereby incorporated by reference in theirentirety). The hydroxyl-terminated self-assembled monolayer (SAM), whichcannot be activated by N-hydroxysuccinimide (NHS) andN-ethyl-N′-(3-diethylaminopropyl) carbodiimide (EDC), was used as aspacer to construct the sensor surface. Second, following activation ofthe terminal carboxylic groups of the mixed SAM with a 1:1 mixture of0.1 M NHS and 0.4 M EDC for 10 min, streptavidin (SA; 200 μg/ml; MPBiomedicals) in 10 mM sodium acetate buffer (pH 5.5) was injected andallowed to covalently couple for 15-20 min followed by PBS rinsing andblocking with 1.0 M ethanolamine hydrochloride (pH 8.5) for 10 min. Thisresulted in a large increase in the SPR wavelength. Because of the closerelationship that exists between electrostatic binding and covalentbinding of SA with carboxylic terminated SAMs, this increase in SPRsignal is a decent estimate of the extent of covalent binding of SA tothe SAMs (Choi et al., “Enhanced Performance of a Surface PlasmonResonance Immunosensor for Detecting Ab-GAD Antibody Based on theModified Self-Assembled Monolayers,” Biosens Bioelectron 21:378-83(2005), which is hereby incorporated by reference in its entirety).Third, following a thorough PBS wash, biotinylated rabbit anti-E. coliantibody (140 μg/ml in PBS; Cortex Biochem) was injected over the SAsurface for 20 min and unbound biotinylated anti-E. coli antibody wasremoved by washing with PBS for 10 min. This resulted in an exponentialincrease in the SPR wavelength that was characteristic of electrostaticbinding between SA and biotin-conjugated proteins. As control, bovineserum albumin (BSA; 140 μg/ml in sodium acetate buffer) was added inplace of anti-E. coli antibody to the SA-coated reference channel of theSPR sensor chip and, as expected, no detectable SPR wavelength shift wasobserved following introduction of BSA or, subsequently, anti-E. coliantibody.

Example 10 Fluorescence Microscopy

For immunofluorescence studies, E. coli cells that had been induced toexpress GFP or ClyA-GFP were washed three times in PBS, incubated at 4°C. overnight with mouse anti-GFP (or anti-polyhistidine) diluted 1:500,pelleted, washed three times with PBS, incubated for 1-hr withrhodamine-labeled goat anti-mouse IgG (Molecular Probes, Carlsbad,Calif.) diluted 1:100, pelleted and washed three more times with PBS.Finally, cells were examined by a Zeiss Axioskop 40 fluorescentmicroscope with Spotflex color digital camera and filter sets for GFP(485 nm for excitation and 505 nm for emission) and Rhodamine (540 nmfor excitation and 600 nm for emission). For fluorescent studies of OMVinteractions with eukaryotic cells, HeLa cells grown on glass coverslipswere washed in OptiMEM (Life Technologies, Carlsbad, Calif.) withoutserum and then treated as described herein. Following treatment, cellswere fixed with 3.7% formalin in PBS, washed three times in PBS,permeabilized in PBS/0.1% Triton X-100, stained with 0.5 mg/mL ethidiumbromide in PBS, and finally washed three times in PBS. Coverslips weremounted onto glass slides using Vectashield Hardset mounting medium(Vector Laboratories, Burlingame, Calif.) prior to wide-fieldepifluorescence analysis. For WGA studies, non-permeabilized cells wereincubated with 1 μg/ml Texas red WGA (Molecular Probes) for 1 h at 4° C.For G_(M1) experiments, ˜150 μg of vesicles were preincubated with 10 μgG_(M1) (Sigma) for 30 min at 25° C.

Example 11 Electron Microscopy

Ultrastructural analysis of vesicles was performed by negative stainingtechnique as described previously (Wai et al., “The Release of OuterMembrane Vesicles from the Strains of Enterotoxigenic Escherichia coli,”Microbiol Immunol 39:451-6 (1995), which is hereby incorporated byreference in its entirety). For immunogold labeling, a 10-μL suspensionof induced E. coli cells was collected, washed and applied to 400-meshFormvar- and carbon-coated copper grids (Electron Microscopy Sciences,Hatfield, Pa.) and incubated for 1 h with anti-GFP diluted 1:500. Cellswere washed with PBS, incubated for 1 h with 25 nm colloidalgold-conjugated goat anti-mouse IgG (Electron Microscopy Sciences)diluted 1:100, washed again, negatively stained with 0.25%phosphotungstic acid (PTA, Electron Microscopy Sciences) with 0.01% BSAin water and viewed using a FEI/Philips Morgagni transmission electronmicroscope.

Example 12 Cytotoxicity Assay

Vesicles were prepared in PBS and total protein in vesicle fractions wasquantified by the Coomassie Plus Assay (Pierce) using BSA proteinstandards. HeLa cells were grown in clear, flat-bottom tissue culturepolystyrene 96-well plates (Costar) at an initial density of 5,000 cellsper well in 200 μL of growth medium. After 24 h, the growth medium wasremoved and replaced with 110 μL of Opti-MEM I® (Invitrogen) serum-freemedium and 40 μL of undiluted (1×; ˜90-150 μg/ml total protein) or 1:1diluted (0.5×; ˜40-60 μg/ml total protein) OMV samples in PBS. Cellswere incubated in the presence of vesicle samples for 4 h; afterwardsthe OMV-containing medium was removed and replaced with 175 μL of phenolred-free growth medium. Following an additional 48 h of incubation, 35μL of CellTiter 96® Aqueous One Solution Cell Proliferation Assayreagent (Promega, Madison, Wis.) was added to the wells. This assay usesthe tetrazolium compound[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS] and the electron coupling reagent phenazinemethosulfate. MTS is chemically reduced by cells into formazan whoseconcentration and optical absorbance at 490 nm provide a measure ofmetabolically active live cells. Samples were incubated for 1 h and theabsorbance was read in a microplate spectrophotometer at 490 nm. Cellviability is reported relative to PBS controls.

Example 13 GFP Co-localizes in Outer Membrane Vesicles When Fused toClyA

Previous studies demonstrated that genetic fusions between E. coli ClyAand reporter proteins such as Bla and GFP were efficiently translocatedacross the cytoplasmic membrane (del Castillo et al., “Secretion of theEscherichia coli K-12 SheA Hemolysin is Independent of its CytolyticActivity,” FEMS Microbiol Lett 204:281-5 (2001) and Galen et al.,“Adaptation of the Endogenous Salmonella enterica serovar TyphiClyA-Encoded Hemolysin for Antigen Export Enhances the Immunogenicity ofAnthrax Protective Antigen Domain 4 Expressed by the AttenuatedLive-Vector Vaccine Strain CVD 908-htrA,” Infect Immun 72:7096-106(2004), which are hereby incorporated by reference in their entirety)and that localization was independent of the position (N- or C-terminus)of ClyA in the fusion protein (del Castillo et al., “Secretion of theEscherichia coli K-12 SheA Hemolysin is Independent of its CytolyticActivity,” FEMS Microbiol Lett 204:281-5 (2001), which is herebyincorporated by reference in its entirety). Separately, Wai andcoworkers demonstrated that ClyA was exported from laboratory strains ofE. coli cells via OMVs composed of outer membrane and periplasm (Wai etal., “Characterization of dominantly negative mutant ClyA cytotoxinproteins in Escherichia coli,” J Bacteriol 185:5491-9 (2003), which ishereby incorporated by reference in its entirety). These same authorsreported that ClyA was significantly enriched in OMVs relative to otherlumenal and membrane-bound OMV proteins (Wai et al., “Vesicle-MediatedExport and Assembly of Pore-Forming Oligomers of theEnterobacterial ClyACytotoxin,” Cell 115:25-35 (2003), which is hereby incorporated byreference in its entirety). Based on these results, it was hypothesizedthat proteins fused to the N- or C-terminus of ClyA would be efficientlyco-localized in OMVs and would retain their native function followingvesicle localization. To test this, fusion constructs between GFP andthe N- or C-terminus of ClyA were generated. Expression of these fusionproteins in the OMV hyper-producing strain JC8031 (Bernadac et al.,“Escherichia coli tol-pal Mutants Form Outer Membrane Vesicles,” JBacteriol 180:4872-8 (1998), which is hereby incorporated by referencein its entirety) followed by purification of vesicles from cell-freeculture supernatants yielded uniform s-MVs (FIG. 1A) with an averagediameter (FIG. 1B) and zeta-potential that were nearly indistinguishablefrom naked OMVs produced from plasmid-free JC8031 cells. This result wasconsistent with earlier findings that vesicle density and size wereunaltered due to the incorporation of a heterologous vesicle protein(Kesty et al., “Incorporation of Heterologous Outer Membrane andPeriplasmic Proteins into Escherichia coli Outer Membrane Vesicles,” JBiol Chem 279:2069-76 (2004), which is hereby incorporated by referencein its entirety). A significant level of ClyA-GFP or GFP-ClyA waslocalized in vesicles whereas unfused GFP expressed alone was notdetected in the s-MV preparations (FIG. 1C). Consistent with earlierstudies of ClyA localization, (Wai et al., “Vesicle-Mediated Export andAssembly of Pore-Forming Oligomers of the Enterobacterial ClyACytotoxin,” Cell 115:25-35 (2003) and Wai et al., “Characterization ofDominantly Negative Mutant ClyA Cytotoxin Proteins in Escherichia coli,”J Bacteriol 185:5491-9 (2003), which are hereby incorporated byreference in their entirety) subcellular fractionation of E. coli cellsrevealed that unfused ClyA accumulated in the cytoplasm, periplasm andOMV fractions (FIG. 1D). Likewise, the addition of GFP as an N- orC-terminal passenger protein resulted in a similar pattern oflocalization (FIG. 1D), although the amount of ClyA fusions in theinsoluble fraction clearly increased compared to unfused ClyA. Bothfusion proteins were fluorescent in the cytoplasm, periplasm, and OMVfractions (FIGS. 1E-F) and the whole cell fluorescence associated withcells expressing ClyA-GFP or GFP-ClyA was nearly as bright as cellsexpressing GFP alone. Taken together, the data clearly indicate that GFPwas compatible with ClyA translocation as chimeras between these twoproteins co-localized in OMVs without significant losses in fluorescenceactivity.

The quality of the fractionation procedure was confirmed by theobservation that endogenously expressed outer membrane protein OmpA wasalways found predominantly in the OMV fraction (shown for cellsexpressing ClyA-GFP, FIG. 1D), consistent with earlier studies, (Wai etal., “Vesicle-mediated Export and Assembly of Pore-Forming Oligomers ofthe Enterobacterial ClyA Cytotoxin,” Cell 115:25-35 (2003), which ishereby incorporated by reference in its entirety) while GroEL was foundexclusively in the cytoplasmic fraction and DsbA in the periplasmicfraction (FIG. 1D). DsbA also accumulated to high levels in the OMVfraction (FIG. 1D). While it is common for periplasmic proteins tobecome entrapped in OMVs, (Horstman et al., “Enterotoxigenic Escherichiacoli Secretes Active Heat-Labile Enterotoxin Via Outer MembraneVesicles,” J Biol Chem 275:12489-96 (2000); McBroom et al., “OuterMembrane Vesicles,” In EcoSal-Escherichia coli and Salmonella: Cellularand Molecular Biology (III, R. C., ed.). ASM Press, Washington, D.C.(2005); Wai et al., “Vesicle-Mediated Export and Assembly ofPore-Forming Oligomers of theEnterobacterial ClyA Cytotoxin,” Cell115:25-35 (2003); and Kesty et al., “Incorporation of Heterologous OuterMembrane and Periplasmic Proteins into Escherichia coli Outer MembraneVesicles,” J Biol Chem 279:2069-76 (2004), which are hereby incorporatedby reference in their entirety) the presence of DsbA was unexpectedbased on the findings of Wai and coworkers who reported that thisprotein was excluded from their OMV fractions (Wai et al.,“Vesicle-Mediated Export and Assembly of Pore-Forming Oligomers oftheEnterobacterial ClyA Cytotoxin,” Cell 115:25-35 (2003), which ishereby incorporated by reference in its entirety). One explanation forthis discrepancy might have been related to the use of the tolRA mutantstrain JC8031, which despite its tendency to secrete copious amounts ofvesicles is also characterized by a leaky outer membrane (Bernadac etal., “Escherichia coli tol-pal Mutants Form Outer Membrane Vesicles,” JBacteriol 180:4872-8 (1998) and McBroom et al., “Outer Membrane VesicleProduction by Escherichia coli is Independent of Membrane Instability,”J Bacteriol 188:5385-92 (2006), which are hereby incorporated byreference in their entirety). However, similar patterns of ClyA-GFP andDsbA localization were observed following subcellular fractionation ofΔnlpI mutant cells (FIG. 1D) that are known to produce relatively largequantities of vesicles but do not exhibit membrane instability (McBroomet al., “Outer Membrane Vesicle Production by Escherichia coli isIndependent of Membrane Instability,” J Bacteriol 188:5385-92 (2006),which is hereby incorporated by reference in its entirety). Thus, theexplanation that is currently favored, based on earlier observations,(Horstman et al., “Enterotoxigenic Escherichia coli Secretes ActiveHeat-Labile Enterotoxin Via Outer Membrane Vesicles,” J Biol Chem275:12489-96 (2000), which is hereby incorporated by reference in itsentirety) is that the use of a different host strain resulted in alteredvesicle protein profiles.

To determine whether ClyA-GFP in the pelleted supernatant was associatedwith intact vesicles or with released outer membrane fragments, fusionprotein co-migration with both periplasmic and outer membrane materialwas tested. To this end, pelleted supernatant from cells expressingClyA-GFP was separated by density gradient centrifugation. Westernblotting and densitometry analysis of the resulting fractions revealed agradient profile for ClyA-GFP that peaked in fractions 6-8 (FIGS. 2A andB), reminiscent of the gradient profile of OMV-associated α-hemolysin(Balsalobre et al., “Release of the Type I Secreted alpha-Haemolysin ViaOuter Membrane Vesicles from Escherichia coli,” Mol Microbiol 59:99-112(2006), which is hereby incorporated by reference in its entirety). Asexpected, the maximal GFP activity was detected in the same fractionsthat contained the ClyA-GFP-enriched OMVs (FIGS. 2B and C) althoughweaker fluorescence could be detected in denser fractions (FIG. 2C). Theouter membrane protein OmpA was similarly enriched in fractions 6-8containing the majority of the ClyA-GFP, but strong bands also appearedin fractions 9 and 10 (FIG. 2A). Interestingly, DsbA was more evenlydistributed between fractions 5-10 (FIG. 2A), indicating co-migrationwith vesicles that included a large portion of ClyA-GFP (fractions 6-8)as well as with vesicles that contained lesser amounts of ClyA-GFP(fractions 5, 9 and 10).

Example 14 ClyA Anchors Correctly Folded GFP to the Outer Surface of E.coli and to the Surface of s-MVs

To determine the topology of the ClyA-GFP and GFP-ClyA chimeras, thesurface accessibility of GFP on whole cells and on vesicles was probed.Previous studies showed that a fraction of the secreted ClyA remainslocated on the bacterial cell surface (Wai et al., “Vesicle-MediatedExport and Assembly of Pore-Forming Oligomers of theEnterobacterial ClyACytotoxin,” Cell 115:25-35 (2003), which is hereby incorporated byreference in its entirety). Likewise, it was observed that both ClyA-GFPand GFP-ClyA were localized to the cell surface as evidenced by theaccessibility of the GFP moiety to cross-reacting antibodies.Specifically, positive immunofluorescent- and immunogold-labeling usinganti-GFP antibodies was detected for JC8031 cells expressing ClyA fusedwith GFP but not in the cases of unfused ClyA or unfused GFP (FIG. 3,shown for unfused GFP and ClyA-GFP).

To determine if the GFP associated with vesicles was similarlyaccessible to cross-reacting antibodies, immunofluorescent labeling ofs-MVs was performed but only a weak immunofluorescent signal abovebackground could be seen in this analysis. This prompted the explorationof a more sensitive and quantitative surface plasmon resonance(SPR)-based strategy for detecting vesicle-associated GFP. For this,biotinylated anti-E. coli antibodies (test channel) and bovine serumalbumin (BSA, reference channel) were coupled to an SPR sensor chipthrough streptavidin binding. To verify that the SPR surface couldcapture the s-MVs, solutions containing varying amounts of intact s-MVsfrom fraction 7 above were introduced, where the initial concentrationof s-MVs in this fraction was 13.5±1.34 μg/μl. Following introduction ofClyA-GFP-containing s-MVs to the SPR, it was immediately noticed thatthe test channel coated with anti-E. coli antibodies but not theBSA-coated reference channel was highly fluorescent (FIG. 4A), evenafter several PBS wash steps, indicating specific capture of the s-MVson the SPR surface. SPR binding revealed concentration-dependent shiftsin the SPR wavelength over a range of s-MV concentrations (0.02-0.70μg/μl) (FIGS. 4B and C). Importantly, there was no measurable change inSPR wavelength upon treatment of surface-captured s-MVs with PBS,indicating that the fluorescent vesicles were stably and tightly boundto the immobilized anti-E. coli antibody. Finally, to determine whetherthe SPR strategy was suitable for detecting OMV-associated antigens,anti-GFP monoclonal antibodies were introduced into the test channelthat contained surface-captured s-MVs displaying active GFP. Specificbinding between anti-GFP antibodies and vesicle-associated GFP wasconfirmed by a marked increase in the SPR wavelength in the test channel(FIG. 4D, black line). Controls were performed where these s-MVs weretreated with non-specific anti-His6× monoclonal antibodies or wheres-MVs displaying unfused ClyA were captured on the SPR surface andtreated with anti-GFP antibodies; both cases resulted in no detectableincrease in the SPR wavelength (shown for anti-His6x treatment ofCly-GFP s-MVs in FIG. 4D, gray line).

Proteinase K (PK) susceptibility assays on ClyA-GFP and GFP-ClyAvesicles were performed to determine whether the immuno-accessible GFPwas protected by the vesicle structure. When s-MVs derived from JC8031cells expressing ClyA-GFP or GFP-ClyA were incubated with PK in theabsence of membrane-disrupting detergent, vesicle-associatedfluorescence was completely abolished (FIG. 5A, shown for ClyA-GFP)suggesting that the majority of the functional GFP was surface exposedand not protected by the vesicle structure. Consistent with this,Western blot analysis confirmed that nearly all of the s-MV-associatedClyA-GFP was degraded to a lower molecular weight anti-GFP or anti-C1yAcross-reacting species upon treatment with PK (FIG. 5B, lanes 1-3 and7-9). Interestingly, a significant amount of PK-resistant material wasobserved following identical treatment of GFP-ClyA s-MVs (FIG. 5B, lanes4-6 and 10-12) that persisted even after incubation with 2-5× higher PKconcentrations for 2× longer durations. Since these PK-treated s-MVswere non-fluorescent but contained a considerable portion ofPK-resistant GFP-ClyA, it was concluded that only a fraction of thefusion localizes with functional GFP tethered outside the OMV while theremainder adopts an inactive conformation that is protected by thevesicle structure. Possible reasons for this include a localizationdefect caused by the relatively high level of expression for GFP-ClyAcompared to ClyA-GFP and/or the apparent instability of the fusion asevidenced by the multiple anti-ClyA cross-reacting bands seen in theabsence of PK (FIG. 5B, lane 10). For both chimeras, completeproteolytic digestion of GFP by PK occurred when membranes weredisrupted by the addition of 1% SDS (FIG. 5B). Control experiments usingpurified, soluble ClyA-GFP showed that the protein was PK-sensitive bothin the presence or absence of the detergent.

Example 15 Periplasmic Disulfide Bond-Forming Machinery is Required forLocalization of ClyA and ClyA Fusions in OMVs

In previous work, ClyA in the periplasm was shown to adopt a monomericconformation owing to an intramolecular disulfide bond formed betweenthe cysteine residues at positions 87 and 285 in the polypeptide (Atkinset al., “Structure-function relationships of a novel bacterial toxin,hemolysin E. The role of alpha G,”J Biol Chem 275:41150-5 (2000), whichis hereby incorporated by reference in its entirety). The presence ofthe disulfide bond was sufficient to prevent oligomerization of ClyA andinactivate its native haemolytic activity. In agreement with thisfinding, Wai et al. reported that DsbA, the enzyme responsible forcatalyzing the formation of disulfide bonds in periplasmic proteins, wasabsent from OMVs containing ClyA and that the absence of DsbA wasnecessary for ClyA to oligomerize into its haemolytic conformation (Waiet al., “Vesicle-Mediated Export and Assembly of Pore-Forming Oligomersof theEnterobacterial ClyA Cytotoxin,” Cell 115:25-35 (2003), which ishereby incorporated by reference in its entirety). Contrary to thefindings of Wai et al., DsbA was co-localized in vesicles containing theClyA fusions in these studies (see FIG. 1 above). Thus, it washypothesized that oxidized, monomeric ClyA might be favorable forefficient cell surface and vesicle localization of fusion proteins. Totest this hypothesis, expression of ClyA-GFP in strain JC8031 and anisogenic dsbA::Kan mutant derived from JC8031 was compared. Western blotanalysis revealed that while both strains accumulated similar amounts ofClyA-GFP in the periplasm, only in cells with DsbA present waslocalization of ClyA-GFP observed (FIG. 5C). This was corroborated by acomplete lack of fluorescence seen for OMVs derived from JC8031dsbA::Kan cells expressing ClyA-GFP (FIG. 5D). Immunofluorescentstaining of these same cells revealed that localization of ClyA-GFP tothe bacterial cell surface was also dependent upon DsbA (FIG. 5D). Itwas surprising to find that, DsbA-dependent vesicle localization wasalso observed for unfused ClyA suggesting that the periplasmic redoxstate is a critical factor in regulating the efficiency of proteinlocalization into vesicles under the conditions tested here.Interestingly, very little, to no, apparent cytotoxicity for naked OMVsfrom plasmid-free JC8031 cells was observed, consistent with earlierstudies, (Wai et al., “Vesicle-Mediated Export and Assembly ofPore-Forming Oligomers of theEnterobacterial ClyA Cytotoxin,” Cell115:25-35 (2003), which is hereby incorporated by reference in itsentirety) and also for s-MVs containing ClyA-His6, ClyA-GFP or GFP-ClyAsuggesting that ClyA in these vesicles was not in itshaemolytically-active, oligomeric conformation.

Example 16 Engineered ClyA-GFP s-MVs are Useful for Visualizing VesicleInteractions with Eukaryotic Cells

Previous studies have shown that vesicles derived from pathogenic E.coli or non-pathogenic E. coli strains can associate with eukaryoticcells (Kesty et al., “Enterotoxigenic Escherichia coli Vesicles TargetToxin Delivery into Mammalian Cells,” EMBO J23:4538-49 (2004) and Kestyet al., “Incorporation of Heterologous Outer Membrane and PeriplasmicProteins into Escherichia coli Outer Membrane Vesicles,” J Biol Chem279:2069-76 (2004), which are hereby incorporated by reference in theirentirety). Thus, whether s-MVs functionalized with ClyA-GFP weresuitable for tracking vesicle association with eukaryotic cells wasinvestigated. Previous attempts at this focused on loading GFP into thelumen of vesicles following its transport into the periplasm by thetwin-arginine translocation (Tat) pathway (Kesty et al., “Incorporationof Heterologous Outer Membrane and Periplasmic Proteins into Escherichiacoli Outer Membrane Vesicles,” J Biol Chem 279:2069-76 (2004), which ishereby incorporated by reference in its entirety). However,GFP-containing OMVs were only weakly fluorescent and could not betracked in host cells by microscopy, likely due to the low yield of GFPtransport to the periplasm via the Tat system. To determine if s-MVsengineered with ClyA-GFP were bright enough for tracking studies,vesicle-host cell co-incubation assays were performed. Punctate greenstaining was observed following a 30 min incubation of HeLa cells with˜150 μg vesicles purified ClyA-GFP s-MVs and the intensity of thisstaining increased as the incubation time between HeLa cells andClyA-GFP s-MVs increased (FIG. 6A). These results imply that vesiclespersisted on the cell surface or had fused directly to the target cellmembrane. To corroborate this, HeLa cells that had been incubated withpurified ClyA-GFP-containing s-MVs were stained with a fluorescentversion of the cell surface marker wheat germ agglutinin (WGA) and thenwashed with PBS. Confocal microscopy revealed that ClyA-GFP OMVsco-localized with WGA on the exterior of the cell. Next, the fate ofClyA-GFP vesicles was explored by examining whether the appearance ofpunctate fluorescence was temperature dependent, a hallmark of cellularinternalization (Kesty et al., “Enterotoxigenic Escherichia coliVesicles Target Toxin Delivery into Mammalian Cells,” EMBO J 23:4538-49(2004) and Pelkmans et al., “Caveolar Endocytosis of Simian Virus 40Reveals a New Two-Step Vesicular-Transport Pathway to the ER,” Nat CellBiol 3:473-83 (2001), which are hereby incorporated by reference intheir entirety). HeLa cells incubated with vesicles containing ClyA-GFPat 4° C. exhibited very low levels of cell-associated fluorescence(compare FIG. 6B). However, when HeLa cells were incubated with ClyA-GFPs-MVs at 4° C. for 3 h and then shifted to 37° C. for 3 hr, strong cellfluorescence was observed (FIG. 6B), leaving open the possibility thatsome s-MVs may be internalized at 37° C. A key factor in endocytosis isganglioside M1 (G_(M1)), which is a eukaryotic cell surface receptor forenterotoxins such as LT and cholera toxin (CT) and is required forendocytosis of LT-containing OMVs derived from non-pathogenic E. coli(Kesty et al., “Enterotoxigenic Escherichia coli Vesicles Target ToxinDelivery into Mammalian Cells,” EMBO J 23:4538-49 (2004), which ishereby incorporated by reference in its entirety). Therefore, whetherthe observed fluorescence of HeLa cells incubated with ClyA-GFP s-MVswas G_(M1)-dependent was tested. Indeed, fluorescence associated withHeLa cells was significantly decreased following incubation withpurified ClyA-GFP s-MVs that had been not been pretreated with G_(M1)(FIG. 6C). Moreover, incubation with G_(M1)-treated vesicles resulted ina small number of large fluorescent clusters and much less punctategreen fluorescence than was observed for HeLa cells incubated withuntreated ClyA-GFP OMVs (FIG. 6C). Thus, it appears that G_(M1) cellsurface receptors may play an important role in mediating interactionsbetween HeLa cells and engineered vesicles. Finally, to assay thecytotoxic effect of vesicles on target cells, an analysis of howcultured HeLa cells were affected by equivalent amounts of differentvesicle preparations was performed. In general, vesicles containingClyA-His6 or ClyA-GFP exhibited virtually no detectable cytotoxicity(FIG. 6D), consistent with a monomoric, DsbA+ conformation (Wai et al.,“Vesicle-Mediated Export and Assembly of Pore-Forming Oligomers oftheEnterobacterial ClyA Cytotoxin,” Cell 115:25-35 (2003), which ishereby incorporated by reference in its entirety) for both ClyA andClyA-GFP in these vesicles.

Example 17 Heterologous Proteins Co-localized in s-MVs via ClyA Retaintheir Activity

To determine whether proteins other than GFP could be fused to ClyAwhile still retaining their function, a series of N- and C-terminalfusions between ClyA and the following enzymes: β-lactamase (Bla),organophosphorus hydrolase (OPH) and β-galactosidase (LacZ), wereproduced. Smilar to what was seen for GFP, ClyA-Bla resulted inlocalization of Bla to the surface of JC8031 cells and vesicles asdetermined using a nitrocefin hydrolysis assay (Table 2).

TABLE 2 ClyA-mediated display of enzymatically active proteins ConstructCell surface activity OMV activity β-lactamase (Bla) [ΔA486/min][ΔA486/min] pBAD18-Kan 0.03 0.02 pΔss-Bla 0.47 0.98 pClyA-Bla 8.53 7.05pBla-ClyA 1.43 1.65 Organophosphorus [U*1000/OD₆₀₀] [U*1000/g totalhydrolase (OPH) protein] pBAD24 0.09 0.04 pOPH 0.36 1.63 pClyA-OPH 11.6281.63 pOPH-ClyA 0.22 8.19 β-galactosidase (LacZ) [ΔA420/min] [ΔA420/min]pBAD24 0.02 0.02 pLacZ 0.02 (7.21)^(†) 0.03 pClyA-LacZ 0.04 (8.33)^(†)0.03 ^(†)Values in parentheses represent activity in the cytoplasmicfraction. All values represent the average of 3 replicate experimentswhere the standard error was <5%.

Since Bla expressed in the cytoplasm was not localized to the cellsurface or vesicles and since nitrocefin is relatively impermeable tothe outer membrane, (Angus et al., “Outer Membrane Permeability inPseudomonas aeruginosa: Comparison of a Wild-type with anAntibiotic-Supersusceptible Mutant,” Antimicrob Agents Chemother21:299-309 (1982) and Good et al., “Antisense PNA Effects in Escherichiacoli are Limited by the Outer-Membrane LPS Layer” Microbiology146:2665-70 (2000), which are hereby incorporated by reference in theirentirety) these data provide strong evidence that the Bla moiety wasexternally localized on cells and vesicles. Similar results wereobtained using the Bla substrate penicillin-G, which also penetrates theouter membrane very poorly (Nikaido et al., “Sensitivity of Escherichiacoli to Various Beta-lactams is Determined by the Interplay of OuterMembrane Permeability and Degradation by Periplasmic beta-lactamases: AQuantitative Predictive Treatment,” Mol Microbiol 1:29-36 (1987), whichis hereby incorporated by reference in its entirety). Interestingly, aswas seen above for the chimeras between ClyA and GFP, fusion of Bla tothe N-terminus of ClyA resulted in a significantly lower level of Blaactivity. Consistent with these results, fusion of ClyA to theN-terminus of the OPH enzyme conferred a significant level of OPHactivity to the surface of cells and vesicles, whereas OPH—ClyA resultedin no measurable surface-associated OPH activity (Table 2). Since theOPH substrate paraoxon used in these experiments is membraneimpermeable, (Richins et al., “Biodegradation of OrganophosphorusPesticides by Surface-Expressed Organophosphorus Hydrolase,” NatBiotechnol 15:984-7 (1997), which is hereby incorporated by reference inits entirety) it was concluded that ClyA-OPH is oriented with OPHexternally bound to both cells and vesicles. Also, since OPH activitydepends upon homodimer formation, (Grimsley et al., “OrganophosphorusHydrolase is a Remarkably Stable Enzyme that Unfolds Through aHomodimeric Intermediate,” Biochemistry 36:14366-74 (1997), which ishereby incorporated by reference in its entirety) ClyA apparentlytethers OPH in a conformation that allows for dimerization with aneighboring ClyA-OPH molecule. Finally, to determine if the ability todisplay multimeric enzymes is a general feature of ClyA-mediated surfaceexposure, ClyA fusions to the homotetrameric LacZ enzyme from E. coliwere constructed (Jacobson et al., “Three-Dimensional Structure ofBeta-galactosidase from E. coli,” Nature 369:761-6 (1994), which ishereby incorporated by reference in its entirety). While expression ofClyA-LacZ resulted in strong cytoplasmic LacZ activity, there was nomeasurable LacZ activity on the surface of cells or their derived s-MVs(Table 2). In fact, there was no LacZ activity in the periplasm of cellsexpressing ClyA-LacZ), consistent with the observation that the normallycytoplasmic LacZ protein contains sequences that hinder transport (Leeet al., “Genetic Studies on the Inability of Beta-galactosidase to beTranslocated Across the Escherichia coli Cytoplasmic Membrane,” JBacteriol 171:4609-16 (1989), which is hereby incorporated by referencein its entirety) usually leading to a misfolded, and thus inactive,protein.

Single-chain antibody fragments (scFv) have been successfully used tocreate artificial immuno liposomes for targeting of these vesicles andtheir payloads to specific cell types (Kontermann, R. E.,“Immunoliposomes for Cancer Therapy,” Curr Opin Mol Ther 8:39-45 (2006),which is hereby incorporated by reference in its entirety). Alongsimilar lines, it was desirable to create bacterial “immuno-MVs” bydisplaying scFv fragments on E. coli-derived vesicles. For theseexperiments, an scFv derived from the 26-10 monoclonal antibody thatbinds with high affinity (K_(D)=0.9±0.2×10-9 M⁻¹) to the cardiacglycoside digoxin (scFv.Dig) was used (Daugherty et al., “QuantitativeAnalysis of the Effect of the Mutation Frequency on the AffinityMaturation of Single Chain Fv antibodies,” Proc Natl Acad Sci USA97:2029-34 (2000) and Francisco et al., “Production andFluorescence-Activated Cell Sorting of Escherichia coli Expressing aFunctional Antibody Fragment on the External Surface,” Proc Natl AcadSci USA 90:10444-8 (1993), which are hereby incorporated by reference intheir entirety). Using a fluorescent conjugate of digoxin (Dig-BODIPY),expression of ClyA-scFv.Dig, but not scFv.Dig alone, resulted in cellsand vesicles that were able to bind the fluorescent probe (FIG. 7A).Since Dig-BODIPY cannot permeate the outer membrane under standardconditions, (Chen et al., “Isolation of High-Affinity Ligand-BindingProteins by Periplasmic Expression with Cytometric Screening (PECS),”Nat Biotechnol 19:537-42 (2001), which is hereby incorporated byreference in its entirety) the detection of Dig-BODIPY binding activityusing intact cells indicates that scFvs were functionally displayed onthe outer cell and vesicle surface. For comparison, cells expressingscFv.Dig fused to the well-characterized Lpp-OmpA hybrid OM anchor(Francisco et al., “Production and Fluorescence-Activated Cell Sortingof Escherichia coli Expressing a Functional Antibody Fragment on theExternal Surface,” Proc Natl Acad Sci USA 90:10444-8 (1993), which ishereby incorporated by reference in its entirety) displayed uniform butnotably weaker cell surface fluorescence and no detectable fluorescenceon OMVs (FIG. 7A) despite the fact that wildtype OmpA localizes in OMVs(Wai et al., “Vesicle-Mediated Export and Assembly of Pore-FormingOligomers of the Enterobacterial ClyA Cytotoxin,” Cell 115:25-35 (2003),which is hereby incorporated by reference in its entirety) (see alsoFIG. 1D above).

Lastly, it was determine whether the capture of Dig-BODIPY byClyA-scFv.Dig could be used as a genetic screen for ClyA localization.Consistent with the fluorescence microscopy results above, labeling ofJC8031 cells expressing ClyA-scFv.Dig with Dig-BODIPY resulted in highlyfluorescent cells as revealed by flow cytometry (FIG. 7B). However, whenClyA-scFv.Dig was expressed in JC8031 dsbA::Kan cells, fluorescence wascompletely abolished. Likewise, when scFv.Dig was expressed alone, as anN-terminal fusion to ClyA (scFv.Dig-ClyA), or as a C-terminal fusion toClyA variants carrying mutations that were previously reported todisrupt translocation (e.g., deletion of 10-147 of the last C-terminalamino acids (Wai et al., “Characterization of Dominantly Negative MutantClyA Cytotoxin Proteins in Escherichia coli,” J Bacteriol 185:5491-9(2003), which is hereby incorporated by reference in its entirety) orsubstitution of the Tyr288 residue with Gly (del Castillo et al.,“Secretion of the Escherichia coli K-12 SheA Hemolysin is Independent ofits Cytolytic Activity,” FEMS Microbiol Lett 204:281-5 (2001), which ishereby incorporated by reference in its entirety)), no measurable cellfluorescence was detected (FIG. 7B).

Discussion of Examples 1-17

This work describes the development and characterization of engineeredsynthetic membrane vesicles (s-MVs) created by genetic fusion of arecombinant polypeptide with the E. coli cytotoxin ClyA. In general, itwas observed that most recombinant polypeptide fusions co-localized withClyA to the bacterial cell surface and into OMVs. Specifically, it wasdemonstrated that direct fusion of Bla, OPH, GFP and anti-digoxin scFvto the C-terminus of ClyA resulted in functional display of each proteinon the surface of E. coli cells and their derived OMVs, giving rise tos-MVs with significantly expanded, non-native functionality (e.g.,fluorescence, antigen binding). Interestingly, fusion of each of theseproteins to the N-terminus of ClyA yielded unpredictable results. Forinstance, scFv.Dig-ClyA exhibited no detectable activity on the surfaceof cells or OMVs while GFP-ClyA resulted in the display of activeprotein. In the latter case, even though a portion of the fusions wasactive, a significant amount of non-fluorescent GFP-ClyA accumulated inOMVs. It was also found that fusion of the enzymes Bla and OPH resultedin little to no activity on cells and OMVs when each enzyme was fused tothe N-terminus of ClyA. These results are consistent with earlierstudies where a 2-fold increase in secretion to the extracellular mediumwas observed for ClyA-Bla versus Bla-ClyA (del Castillo et al.,“Secretion of the Escherichia coli K-12 SheA Hemolysin is Independent ofits Cytolytic Activity,” FEMS Microbiol Lett 204:281-5 (2001), which ishereby incorporated by reference in its entirety). At present, it is notknown why N-terminal fusions to ClyA were inconsistent whereasC-terminal fusions invariably yielded well-displayed proteins thatretain their biological function. These results might be explained byinspection of the medium-resolution structure of membrane-bound ClyAshowing that the C-terminus of ClyA is embedded deeper within membranes(i.e., closer to the outer surface) than the N-terminal portion, whichoccurs close to the periplasmic side of the outer membrane (Eifler etal., “Cytotoxin ClyA from Escherichia coli Assembles to a 13-meric PoreIndependent of its Redox-State,” EMBO J 25:2652-61 (2006) and Tzokov etal., “Structure of the hemolysin E (HlyE, ClyA, SheA) channel in itsmembrane-bound form,” J Biol Chem 281:23042-9 (2006), which is herebyincorporated by reference in its entirety). According to this model,fusions to the C-terminus of ClyA are closer to the outer surface andmore likely to extend into the extracellular environment, especiallywith the addition of a flexible 5-residue Gly linker that was includedin all of fusions.

Based on its relative plasticity as a fusion partner, ClyA can serve asa useful tethering module for: (1) dissecting the complete ClyAtranslocation pathway from bacteria to target host cells; and (2)biotechnological applications that rely on cell or OMV surface display,such as affinity maturation of antibody fragments and vaccine adjuvantdevelopment (Daugherty et al., “Quantitative Analysis of the Effect ofthe Mutation Frequency on the Affinity Maturation of Single Chain FvAntibodies,” Proc Natl Acad Sci USA 97:2029-34 (2000); Francisco et al.,“Production and Fluorescence-Activated Cell Sorting of Escherichia coliExpressing a Functional Antibody Fragment on the External Surface,” ProcNatl Acad Sci USA 90:10444-8 (1993); Chen et al., “Cell-Surface Displayof Heterologous Proteins: From High-Throughput Screening toEnvironmental Applications,” Biotechnol Bioeng 79:496-503 (2002); andGeorgiou et al., “Display of Heterologous Proteins on the Surface ofMicroorganisms: From the Screening of Combinatorial Libraries to LiveRecombinant Vaccines,” Nat Biotechnol 15:29-34 (1997), which are herebyincorporated by reference in their entirety). For instance, with respectto genetic analysis, expression of ClyA-GFP and ClyA-scFv.Dig bothproved capable of reporting the localization of ClyA to the bacterialcell surface and into OMVs and revealed an essential role for DsbA inthe translocation process. A simple model for ClyA assembly in vesiclesproposed by Uhlin and coworkers, and supported by these findings, isthat ClyA oligomerization and membrane insertion is governed by theredox state of ClyA (Wai et al., “Vesicle-Mediated Export and Assemblyof Pore-Forming Oligomers of the Enterobacterial ClyA Cytotoxin,” Cell115:25-35 (2003), which is hereby incorporated by reference in itsentirety). Here, however, it was shown that a more reducing environmentin the periplasm, such as occurs when DsbA is absent, favors thelocalization of ClyA and ClyA fusions into vesicles. While theinvolvement of DsbA appears to be via direct oxidation of ClyA, (Wai etal., “Vesicle-Mediated Export and Assembly of Pore-Forming Oligomers ofthe Enterobacterial ClyA Cytotoxin,” Cell 115:25-35 (2003), which ishereby incorporated by reference in its entirety) the possibility thatDsbA is responsible for the oxidation of a periplasmic or membranecomponent(s) that mediate the localization of ClyA and/or the formationof OMVs cannot be ruled out.

With respect to biotechnology, the ability of ClyA to anchor andfunctionally display a variety of different prokaryotic and eukaryoticproteins on the surface of bacterial cells and vesicles should be usefulfor numerous applications. First, ClyA exhibits properties that make itan ideal carrier protein for bacterial cell surface display of peptidesand proteins and compares favorably with the well-established Lpp-OmpAsurface anchor as evidenced by scFv binding of fluorescently taggeddigoxin. Second, by judicious selection and display of specific scFvfragments on OMVs, one might be able to redirect these engineered“immuno-MVs” to specific cell types, or otherwise engineer thehost-vesicle interaction, in order to achieve a desired therapeutic orimmunological response. Along these lines, these data provideencouraging data that engineered vesicles retain their characteristicinteractions with mammalian cells (Kesty et al., “EnterotoxigenicEscherichia coli Vesicles Target Toxin Delivery into Mammalian Cells,”.EMBO J 23:4538-49 (2004) and Kesty et al., “Incorporation ofHeterologous Outer Membrane and Periplasmic Proteins into Escherichiacoli Outer Membrane Vesicles,” J Biol Chem 279:2069-76 (2004), which ishereby incorporated by reference in its entirety) and are almostcompletely non-cytotoxic. It is also noteworthy that expression of allClyA fusions in this study had no measurable effect on the growth rateof bacterial cells relative to cells expressing unfused ClyA or emptyvector controls, thus appreciable yields of cells or their derived OMVsshould be attainable for any OMV-based application.

Example 18 Plasmid Construction

Plasmids for ClyA, GFP, and ClyA-GFP were constructed with C-terminal6×-histidine tags to facilitate purification of the protein products.The plasmids pClyA-His6, encoding the E. coli gene clyA fused to the 5′end of a 6×-histidine tag, and pClyA-GFP, encoding clyA fused to the 5′end of gfp-mut2 (26) are described supra in Example 1. To constructpClyA-GFP-His6, a ˜1.7 kb fragment was amplified by polymerase chainreaction (PCR) with plasmid pClyA-GFP as template using primers(5′-TCGCAACTCTCTACTGTTTC-3′) (SEQ ID NO:13) and (5 ′-GCGATGAAGCTTTTAATGGTGATGGTGATGATGTTTGTATAGTTCATCCATGCC-3′) (SEQ ID NO:14). Theresulting product was cloned in the XbaI and HindIII sites of pBAD18-Cm(Guzman et al., “Tight Regulation, Modulation, and High-Level Expressionby Vectors Containing the Arabinose P-Bad Promoter,” J Bacteriol177:4121-4130 (1995), which is hereby incorporated by reference in itsentirety). For construction of pGFP-His6, a ˜700 by fragment wasamplified by PCR with plasmid pClyA-GFP as template and using primers(5′-GCGATGGAATTCGAGCTCTTAAAGAGGAGAAAGGTCATGAGTAAAGGA GAAGAACTTTT-3′)(SEQ ID NO:15) and (5′-GCGATGAAGCTTTTAATGGTGATGGTGATGATGTTTGTATAGTTCATCCATGCC-3′) (SEQ ID NO:16). The amplification product wascloned into pBAD18-Cm using the Sad and HindIII restriction sites. DNAconstructs were verified by automated dideoxy chain-terminationsequencing. Plasmids were transformed into E. coli DH5a and selected inLB medium containing chloramphenicol.

Example 19 Recombinant Protein Purification

Cultures of E. coli DH5α were grown in 100 mL of LB medium containingchloramphenicol. Protein expression was induced by addition ofL-arabinose to a final concentration of 0.2% once the OD₆₀₀ reachedapproximately 0.5. Bacterial cultures were harvested four hours afterinduction and the polyhistidine-tagged proteins were purified byimmobilized-metal affinity chromatography (Ni-NTA Agarose, Qiagen)according to the manufacturer's instructions. The proteins were elutedfrom the affinity resin with 200 mM imidazole in a buffer containing 50mM NaH₂PO₄, 300 mM NaCl, pH 8.0, and subsequently desalted intophosphate buffered saline using PD-10 size exclusion chromatographycolumns (Amersham Biosciences).

Example 20 Preparation of Outer Membrane Vesicles

Outer membrane vesicles were purified in accordance with a previouslyestablished procedure (Kolling, G L et al., “Export of Virulence Genesand Shiga Toxin by Membrane Vesicles of Escherichia coli 0157: H7,” ApplEnviron Microbiol 65:1843-1848 (1999), which is hereby incorporated byreference in its entirety). Plasmids pClyA-GFP-His6 and pBAD18-Cm weretransformed into E. coli vesicle-overproducing strain JC8031 (Bernadac,A et al., “Escherichia coli tol-pal Mutants Form Outer MembraneVesicles,” J Bacteriol 180:4872-4878 (1998), which is herebyincorporated by reference in its entirety), and selected inLB-chloramphenicol medium. Flasks containing 250 mL of medium wereinoculated with overnight culture and allowed to grow until the OD₆₀₀reached approximately 0.5. Protein expression was induced by addition ofL-arabinose to a final concentration of 0.2%. Cell-free culturesupernatants were collected 12 hours after induction and filteredthrough a 0.45 μm vacuum filter. Vesicles were isolated byultracentrifugation (Beckman-Coulter Ti SW28 rotor, 141,000 g, 3 hours,4° C.) and resuspended in phosphate buffered saline (PBS).

Example 21 Protein Analyses

Protein concentrations in OMV and purified recombinant proteinpreparations were quantified by the bicinchoninic acid assay (BCAProtein Assay, Pierce), with bovine serum albumin as the proteinstandard. Fluorescence activity of GFP in protein or OMV samples wasmeasured in a microplate spectrofluorometer (Gemini EM, MolecularDevices) using excitation and emission wavelengths of 481 nm and 507 nm,respectively (Cormack, B P et al., “FACS-Optimized Mutants of the GreenFluorescent Protein (GFP),” Gene 173:33-38 (1996), which is herebyincorporated by reference in its entirety). For SDS-PAGE, samples wereprepared in sample loading buffer containing β-mercaptoethanol andheated at 100° C. for 5 minutes prior to electrophoresis on 10%polyacrylamide gels. Proteins were transferred to polyvinylidinedifluoride membranes for Western blot analysis and probed withmonoclonal mouse anti-GFP IgG (Invitrogen, 1:2,000) or monoclonal mouseanti-polyHistidine IgG (1:3000, Sigma) primary antibodies, andhorseradish peroxidase conjugated goat anti-mouse IgG (1:10,000, JacksonImmunoresearch) secondary antibody. Membranes were developed byautoradiography with ECL detection reagents (Pierce).

Example 22 Liquid Hemolysis Assay

The hemolytic activity of ClyA and ClyA-GFP was measured by a liquidhemolysis assay adopted from Rowe and Welch (Rowe et al., “Assays ofHemolytic Toxins,” Methods Enzymol 235:657-667 (1994), which is herebyincorporated by reference in its entirety). Sheep erythrocytes (BectonDickinson) were washed and diluted 1:100 in PBS. Aliquots of washederythrocytes were transferred to microcentrifuge tubes and ClyA orClyA-GFP was added to the appropriate concentration in a final volume of1 mL in PBS. The samples were incubated at 37° C. for 30 min with gentlerotation. Cells and debris were sedimented in a microcentrifuge (4,000rcf, 1.5 min), and the released hemoglobin in the supernatant wasquantified by spectrophotometric detection at a wavelength of 540 nm.Hemolysis activity was reported relative to erythrocytes lysed indeionized water for 30 minutes at 37° C., which was considered 100%lysis.

Example 23 Dynamic Light Scattering

Dynamic light scattering measurements were performed with the NanosizerNano ZS (Malvern Instruments), using Dispersion Technology Softwareversion 4.20 for data acquisition and analysis. OMV samples contained 60ug/mL total protein in 1 mL of PBS. The refractive index and viscosityof water were used as parameter inputs.

Example 24 Microscopy

For negative staining electron microscopy, vesicles were stained with 2%uranyl acetate on 400-mesh Formar/carbon-coated copper grids and viewedin a FEI Tecnai F20 transmission electron microscope. For fluorescencemicroscopy, vesicles were placed on a glass slide, sealed with a coverslip, and examined using an Olympus BX41 microscope with GFP filter set.

Example 25 Lipopolysaccharide Detection

Bacterial lipopolysaccharide (LPS) concentrations were determined bymeasuring the presence of 2-keto-3-deoxyoctonate (KDO) according to apreviously described colorimetric assay (Karkhanis et al., “New andImproved Microassay to Determine 2-Keto-3-Deoxyoctonate inLipopolysaccharide of Gram-Negative Bacteria,” Anal Biochem 85:595-601(1978) and Herlax et al., “Role of Lipopolysaccharide on the Structureand Function of alpha-Hemolysin from Escherichia coli,” Chem Phys Lipids135:107-115 (2005), which are hereby incorporated by reference in theirentirety). KDO forms part of the oligosaccharide core of the bacterialLPS molecule, with E. coli LPS containing two reactive KDO moieties permolecule (Lee et al., “Quantification of Bacterial Lipopolysaccharidesby the Purpald Assay: Measuring Formaldehyde Generated from2-keto-3-deoxyoctonate and Heptose at the Inner Core by PeriodateOxidation,” Anal Biochem 267:161-168 (1999), which is herebyincorporated by reference in its entirety). OMV samples (45 μL) in PBSwere combined with 0.2 N H₂SO₄ (5 μL) and heated at 100° C. for 20minutes. The samples were cooled to room temperature for five minutesand then 25 μL of 0.04 M NaIO₄ was added to the mixture. Following 20minutes of incubation at room temperature, 2% NaAsO₂ (65 μL) was addedto the sample tubes and vortexed until the characteristic yellow colordisappeared. Thiobarbituric acid (0.3%, 250 μL) was added and thesamples were returned to the boiling water bath for 10 minutes, followedby immediate addition of dimethyl sulfoxide (125 μL). The samples werecooled to room temperature for five minutes and the absorbance wasquantified at 550 nm in a microplate spectrophotometer. Calibrationstandards were prepared from KDO ammonium salt (Sigma-Aldrich).

Example 26 Immunization

Six groups of five BALB/c mice (Charles River Laboratories) each wereimmunized subcutaneously with 100 μL of PBS containing purified proteinor OMV preparations as described. The six treatment groups wereimmunized with: 2.5 μg GFP (group I); 2.5 μg ClyA (group II); 5 μgClyA-GFP (group III); 2.5 μg ClyA mixed with 2.5 μg GFP (group IV); 2.5μg ClyA-GFP mixed with empty OMV (group V); and recombinant OMVequivalent to 2.5 μg ClyA-GFP (group VI). Two doses of vaccine wereadministered four weeks apart. Blood was collected from the mandibularsinus immediately prior to and two weeks following the firstimmunization, immediately prior to the booster dose, and at weeklyintervals thereafter. The methods used in these animal studies wereapproved by the Institutional Animal Care and Use Committee at CornellUniversity.

Example 27 Enzyme-linked Immunosorbent Assay (ELISA)

GFP-reactive antibodies were measured by indirect ELISA. Polystyrenemicrotiter 96-well plates (Maxisorp, Nunc Nalgene) were coated with GFP(5 μg/mL in carbonate buffer, pH 9.6) and incubated overnight at 4° C.Plates were blocked with 3% nonfat dry milk (Bio-Rad) in PBS containing0.05% Tween-20 (PBST). Samples were serially diluted two fold inblocking buffer, in a range of 1:200 to 1:204,800, added to the wells,and incubated for 1.5 hours in a humidified chamber at 37° C. Plateswere washed six times with PBST and then horseradishperoxidase-conjugated goat anti-mouse IgG (1:5,000, JacksonImmunoresearch), was added to the wells for 1 hour at 37° C. Followingsix additional washes with PBST, 3,3′,5,5′ tetramethylbenzidinesubstrate (1-Step Turbo TMB, Pierce) was added and the enzyme reactionproceeded for 20 minutes. The reaction was stopped with 2 M H₂SO₄. Theabsorbance was quantified in a microplate spectrophotometer at awavelength of 450 nm. Statistical significance between treatment groupswas determined by the non-parametric Wilcoxan rank-sum test, with p<0.05considered significant.

Example 28 Fusion of Model Antigen GFP to ClyA Results in Expression ofa 61 kDa Chimeric Protein that Retains the Native Activities of itsComponents

To verify the expression of ClyA, GFP, and ClyA-GFP, proteins werepurified by immobilized metal affinity chromatography from E. colicultures and examined by Western blotting with anti-polyhistidineantibodies. FIG. 8A shows protein bands at 27 kDa corresponding to GFP,at 34 kDa corresponding to ClyA, and at 61 kDa corresponding to theexpected molecular weight of ClyA-GFP. The ClyA-GFP fusion was furtherexamined for the characteristic hemolytic and fluorescence activities ofits constituent proteins. FIG. 8B shows that the degree of hemolysis ofsheep erythrocytes increases with increasing concentration of both ClyAand ClyA-GFP, with ClyA-GFP exhibiting lower hemolysis activity thannative ClyA at all tested concentrations. Similarly, fluorescenceintensity measurements of ClyA-GFP indicate an increase in fluorescenceintensity with increasing concentration, but diminished relative to freeGFP (FIG. 8C). Together, these data show that the intrinsic hemolysisand fluorescence activities of ClyA and GFP, respectively, are retainedwhen the two proteins are fused together as ClyA-GFP, albeit to a lesserdegree than the free proteins likely due to protein proximity.

Example 29 ClyA-GFP is Secreted to the Extracellular Medium of E. coliCultures in Outer Membrane Vesicles

Outer membrane vesicles were prepared from vesicle hyper-producing E.coli strain JC8031 transformed with plasmid pClyA-GFP-His6 (recombinantOMV) or the empty pBAD18-Cm cloning vector (empty OMV). Electronmicroscopy shows the spherical bilayered structure of OMV (FIG. 9A), isapproximately 100 nm in diameter. ClyA-GFP fluorescence was observed inassociation with recombinant OMV (FIG. 9B), a finding confirmed byWestern blotting with anti-GFP antibodies (FIG. 9C). Fluorescenceintensity measurements and SDS-PAGE gel band densitometry indicate thatClyA-GFP comprises approximately 5% of the total protein content in OMV.Since expression of clyA in E. coli is strongly repressed under normallaboratory conditions (Westermark et al., “Silencing and Activation ofClyA Cytotoxin Expression in Escherichia coli,” J Bacteriol182:6347-6357 (2000), which is hereby incorporated by reference in itsentirety), no free ClyA was detected in recombinant or empty outermembrane vesicles. Consistent with earlier observations (Kim et al.,“Engineered Bacterial Outer Membrane Vesicles with EnhancedFunctionality,” J Mol Biol in press: doi:10.1016/j.jmb.2008.1003.1076(2008), which is hereby incorporated by reference in its entirety), theassociation of ClyA-GFP recombinant OMV has no apparent effect on theaverage diameter of vesicles (FIG. 9D). Bacterial lipopolysaccharide(LPS) content in empty and recombinant OMV was measured via acolorimetric assay to detect 2-keto-3-deoxyoctonate (KDO), a core sugarcomponent of LPS. The KDO assay indicates that recombinant OMV contain aslightly higher concentration of LPS, normalized by total proteincontent, than empty OMV (FIG. 9E).

Example 30 The Immunogenicity of GFP in Mice is Significantly Enhancedwhen Administered in Fusion with ClyA

A green fluorescent protein (GFP) model weak antigen was fused to theC-terminus of ClyA to study the immunostimulatory effect of ClyA-antigenfusions in immunized mice. Subcutaneous immunization of BALB/c mice withClyA-GFP elicited GFP-reactive antibody responses that weresignificantly higher than immunization with ClyA mixed with GFP (FIG.10). A GFP-specific IgG response was detected beginning two weeks afterpriming in mice immunized with ClyA-GFP; this response was augmented byadministration of the booster, and sustained for upwards of four weeksfollowing the booster. No detectable anti-GFP IgG antibodies wereobserved until after boosting in any other treatment groups.Interestingly, immunization with GFP elicited little to no detectableresponse at any time during the study period, while in two miceimmunization with ClyA alone triggered fluctuating levels of GFPcross-reactive antibody species following the booster. Antibody titersin the ClyA-GFP immunization group (group III) were significantly higher(p<0.05) than antibody titers in the unfused protein componentstreatment group (group IV) beginning at day 14 of the study and remainedsignificantly higher through the conclusion of the study. GFPcross-reactive antibody levels in the treatment group immunized withClyA and GFP separately (group IV) were statistically similar to theantibody levels generated by ClyA immunization alone (group II) at alltime points throughout the study.

Example 31 ClyA-GFP in an OMV Vaccine Formulation Retains itsImmunogenicity in Mice While Bypassing Intensive Protein PurificationProcesses

To examine the immunogenicity of ClyA-GFP secreted from whole E. colicells in the form of outer membrane vesicles, BALB/c mice were immunizedwith empty OMV mixed with ClyA-GFP or with recombinant outer membranevesicles containing the ClyA-GFP fusion (FIG. 11). The effective dose ofClyA-GFP in the OMV formulations (2.5 μg) was half the amount used inthe purified protein immunizations in an effort to observe ifassociation with OMV contributes any additional immune stimulatingeffect. Immunization with empty OMV combined with purified ClyA-GFP(group V) resulted in a GFP-specific response beginning at two weeksfollowing priming and continuing for four weeks after the booster.Subcutaneous immunization with ClyA-GFP recombinant OMV (group IV)elicited similar GFP-reactive IgG responses starting two weeks followingthe initial dose that then markedly increased following immunizationwith the booster at day 28. At all time points throughout the study,antibody titers between group V and group VI remained statisticallyequivalent. Additionally, titers in OMV-immunized mice (groups V and VI)were statistically equivalent to those in group III with the exceptionsof on day 56 for group V and on day 35 for group VI, when titers weresignificantly higher in group III (p<0.05).

Discussion of Examples 18-31

Vaccination remains one of the most cost-effective strategies forpreventing infectious disease (Levine et al., “New Generation Vaccines,3rd Edition,” eds. (Marcel Dekker, Inc., New York) (2004), which ishereby incorporated by reference in its entirety). The safety of proteinsubunit vaccines over whole organisms makes them particularly attractivefor administration to wide swaths of the human population, healthy andimmunocompromised individuals alike. A major limiting factor in thefurther development of subunit vaccines, however, remains the poorimmunogenicity of purified antigens alone. Despite advances in adjuvantresearch, the only compounds currently licensed for human use in NorthAmerica remains aluminum salts that have potential to cause inflammatoryor allergic reactions (Gupta R K, “Aluminum Compounds as VaccineAdjuvants,” Adv Drug Delivery Rev 32:155-172 (1998), which is herebyincorporated by reference in its entirety). Although particulate antigendelivery systems have emerged as promising strategies for antigendelivery, the added steps of loading or adsorbing purified antigens withparticles on a large manufacturing scale can rapidly render thesesystems cost-prohibitive, a consideration that is especially relevant tovaccines for diseases prevalent in the developing world.

Owing largely to their size, plasticity, and demonstrated safety profilein humans, OMV are attractive vehicles for vaccine delivery. In ademonstration of the remarkable tunability of OMV, heterologous proteinsfused with the native bacterial protein ClyA are efficiently transportedin their native functional forms to vesicles (Kim et al., “EngineeredBacterial Outer Membrane Vesicles with Enhanced Functionality,” J MolBiol in press: doi:10.1016/j jmb.2008.1003.1076 (2008), which is herebyincorporated by reference in its entirety). Previous work also points tothe utility of ClyA in enhancing the immunogenicity of foreign antigenswhen secreted from a live attenuated Salmonella vector, suggesting thepossibility that antigens may be exported from live vectors in OMV(Galen et al., “Adaptation of the Endogenous Salmonella enterica serovartyphi ClyA-Encoded Hemolysin for Antigen Export Enhances theImmunogenicity of Anthrax Protective Antigen Domain 4 Expressed by theAttenuated Live-Vector Vaccine Strain CVD 908-htrA,” Infect Immun72:7096-7106 (2004), which is hereby incorporated by reference in itsentirety).

This work demonstrates that recombinant OMV are a potent adjuvant andcarrier system for poorly immunogenic proteins. Specifically, arecombinant OMV vaccine containing ClyA fused with a model greenfluorescent protein (GFP) foreign antigen elicited strong GFP-specifichumoral responses in immunized mice. These results introduce recombinantOMV vaccines as a versatile strategy for potentially delivering a hostof poorly immunogenic proteins. An important characteristic of OMV isthat they are isolated by a facile ultracentrifugation, effectivelyeliminating costly purification or formulation processes connected withother particulate delivery systems and traditional protein subunitvaccines.

Leveraging the novel secretory behavior of ClyA, a recombinant OMVvaccine containing the model GFP antigen was constructed by fusing GFPwith the C-terminus of ClyA. Consistent with previously observed results(Kim et al., “Engineered Bacterial Outer Membrane Vesicles with EnhancedFunctionality,” J Mol Biol in press: doi:10.1016/j jmb.2008.1003.1076(2008), which is hereby incorporated by reference in its entirety),purification of the engineered ClyA-GFP fusion protein from E. coliculture resulted in a 61 kDa product (FIG. 8A). Functionality assayswere performed to verify that ClyA-GFP in fusion retained the nativebiological activity of ClyA and GFP. The specific fluorescence andhemolysis activity of ClyA-GFP under the specified assay conditions wasdiminished relative to the unfused constituent proteins (FIGS. 8B and8C), which is similar to a previous observation made with other ClyAfusion partners (del Castillo et al., “Secretion of the Escherichia coliK-12 SheA Hemolysin is Independent of its Cytolytic Activity,” FEMSMicrobiol Lett 204:281-285 (2001), which is hereby incorporated byreference in its entirety). This retention of protein activity showsthat proteins fused with ClyA maintain their conformations and indicatesthat ClyA may be an amenable carrier for both linear epitopes andconformational antigens.

Recombinant OMV were purified from non-pathogenic E. coli and examinedfor the presence of ClyA-GFP and LPS (FIG. 9A). Western blotting withanti-GFP antibodies verified the presence of ClyA-GFP in OMV;encapsulation of the active fusion protein in vesicles occurs throughautomatic secretion and thus requires no a priori antigen purification,unlike other particulate delivery systems. Intriguingly, recombinant OMVcontained higher levels of LPS (when normalized with protein content)than did empty vesicles. Because LPS can be counterproductive invaccines, future work may require depletion of LPS from vesicles, as inthe case of the previously described meningitis vaccines (Claassen etal., “Production, Characterization and Control of a Neisseriameningitidis Hexavalent Class 1 Outer Membrane Protein VontainingVesicle Vaccine,” Vaccine 14:1001-1008 (1996), which is herebyincorporated by reference in its entirety).

The results in FIG. 10, depicting antibody titers in mice immunized withpurified proteins, show ClyA to be an effective carrier protein forenhancing the immunogenicity of GFP in mice. The immunogenicity of ClyAfusion proteins was first shown in a study by Galen et al (Galen et al.,“Adaptation of the Endogenous Salmonella enterica serovar typhiClyA-Encoded Hemolysin for Antigen Export Enhances the Immunogenicity ofAnthrax Protective Antigen Domain 4 Expressed by the AttenuatedLive-Vector Vaccine Strain CVD 908-htrA,” Infect Immun 72:7096-7106(2004), which is hereby incorporated by reference in its entirety),where the observed enhancement in the immune response to the antigenswas postulated to be largely dependent on export from a live Salmonellavector. Here, the results show that antigen fusion with ClyA is directlyresponsible for enhanced immunogenicity. As a bacterial cytolyticprotein from enterobacteria (Eifler et al., “Cytotoxin ClyA fromEscherichia coli Assembles to a 13-meric Pore Independent of itsRedox-State,” Embo Journal 25:2652-2661 (2006), which is herebyincorporated by reference in its entirety), ClyA joins the ranks ofother known toxins with adjuvant properties such as diphtheria toxin orheat-labile enterotoxin (Lavelle et al., “Vaccine Adjuvants:Immunological and Clinical Principles,” eds. Hackett C J & Harn D A(Humana Press, Totowa, N.J.), pp. 111-154 (2006), which is herebyincorporated by reference in its entirety). While no pathologicaleffects of were observed in mice immunized with ClyA in this study,detoxification of ClyA through mutation, truncation, or chemical methodsmay attenuate any possible toxicity while retaining its immunomodulatorycapabilities.

Unlike other known bacterial toxin adjuvants, however, enhancement inthe immune response to GFP was not observed when mice were immunizedwith a mixture of unfused ClyA and GFP. While the specific mechanismsthat drive the immunostimulatory behavior of ClyA remain unclear,physical or chemical conjugation to protein carriers has frequentlyimproved the immunogenicity of otherwise poorly immunogenic antigens byincreasing the antigen size or improving antigen organization tooptimize the B cell response (Wu et al., “Sustained High-Titer AntibodyResponses Induced by Conjugating a Malarial Vaccine Candidate toOuter-Membrane Protein Complex,” Proc Nat Acad Sci USA 103:18243-18248(2006); Koser et al., “Rabies Virus Nucleoprotein as a Carrier forForeign Antigens,” Proc Nat Acad Sci USA 101:9405-9410 (2004); andBachmann et al., “The Influence of Antigen Organization on B-CellResponsiveness,” Science 262:1448-1451 (1993), which are herebyincorporated by reference in their entirety). In general, the abilityfor antigens to be presented in the proper structure to activate B cellproliferation requires efficient antigen processing by antigenpresenting cells and co-stimulation by T-cells. Further studies on ClyAand its interaction with the immune system are necessary to more fullycharacterize the immune enhancing activity of ClyA.

To examine the humoral immune response to ClyA-GFP when delivered inOMV, mice were immunized with empty OMV mixed with purified ClyA-GFP(group V), or with the recombinant OMV formulation containing ClyA-GFP(FIG. 11, group VI). The results show that the immunostimulatory effectof ClyA-GFP is retained in vesicles. The kinetics of the antibodyresponse to recombinant OMV appears to occur slightly more slowly thanthe response to purified ClyA-GFP. On day 35 of the study, anti-GFPtiters in the recombinant OMV group (FIG. 11, group VI) were lower thanin the group immunized with purified ClyA-GFP (FIG. 10, group III),however the titers in these two treatment groups are statistically equalto each other beginning at day 42. These results with recombinant OMVare remarkable especially when taking into account that the effectivedose of the ClyA-GFP fusion in the OMV formulations were half that ofthe purified protein treatments. While the decreased dosage may be afactor in the delayed response to ClyA-GFP in mice immunized withrecombinant OMV over the purified protein, it may also be possible thatOMV contributes additional immunostimulatory activity.

Comparison between the empty and recombinant OMV treatments (FIG. 11)suggests that anti-GFP titers are largely independent of the directassociation of ClyA-GFP with OMV. There remains the possibility,however, that non-specific association of purified ClyA-GFP with blankOMV may make the two OMV populations virtually indistinguishable to theimmune system. Association of ClyA-GFP with vesicles does appear,however, to result in a more prolonged antibody response. At day 56, theantibody titers in group V (empty OMV with ClyA-GFP) are statisticallydecreased relative to group III (purified ClyA-GFP), while the titers inthe recombinant OMV treatment group exhibited no such decrease. Similarto other particulate delivery systems, encapsulation of antigens withinOMV may protect antigens from protease degradation in vivo, resulting inmore prolonged B-cell activation.

Recombinant OMV are unique particulate vaccine delivery vectors withadjuvant and carrier activity that combine features borrowed from bothsynthetic and living antigen delivery systems. These vesicles arenon-replicating entities that are able to avoid the potential safetyconcerns associated with attenuated living bacteria. As nanoscalespherical structures comprising mainly protein and lipid, OMV may sharesome structural or compositional similarity with liposome or proteosomecarriers, but do not require extensive formulation. In perhaps the mostsignificant departure from particulate systems or traditional subunitvaccines, recombinant OMV do not require the a priori purification ofthe antigen. The intrinsic ability of ClyA-antigen fusions to betransported to OMV, while retaining biological activity, removesexpensive and often problematic protein purification operations. Theseadvantages in processing and functionality, together with the additionalpossibilities of further engineering lumenal or surface vesicle featuresdemonstrate that OMV are a uniquely tunable platform for the delivery ofpoorly immunogenic antigens for vaccines against infectious disease.

As new disease threats arise and humanitarian needs become ever moreurgent, attention continues to focus on the development of vaccines tosafely induce protective immune responses to pathogens that causedebilitating disease throughout the world. This work illuminates thepotential of recombinant OMV technology to overcome the significantsafety and economic limitations that often arise in the course ofvaccine development. A model green fluorescent protein fused with thebacterial hemolysin ClyA was secreted in outer membrane vesicles whilemaintaining the biological activity of both components. Theserecombinant OMV were administered to mice and found to be highlyimmunogenic, eliciting high titers against GFP while immunization withGFP alone failed to elicit any significant humoral response. Combiningadjuvant and carrier activity, recombinant OMV enhances the response toan otherwise poorly immunogenic antigen and circumvents the proteinpurification requirements of traditional subunit vaccines andparticulate antigen delivery modalities.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. A method of displaying a protein on a cell surface, said methodcomprising: providing either a fusion protein comprising at least aportion of a ClyA protein and at least a portion of a second proteincoupled to said ClyA protein or a nucleic acid construct encoding thefusion protein and administering to a cell the fusion protein or thenucleic acid construct under conditions effective to display the fusionprotein on a surface of the cell.
 2. The method of claim 1, wherein thesecond protein is a marker protein.
 3. The method of claim 1, whereinthe marker protein is a fluorescent protein.
 4. The method of claim 1,wherein the second protein is a ligand binding protein.
 5. The method ofclaim 4, wherein the ligand bind protein is selected from the groupconsisting of high affinity antibody binding fragments, single-chain Fvantibody fragments, nanobodies, fluorobodies, aptamers, biotin-bindingproteins, lipid-binding proteins, periplasmic binding proteins, lectins,serum albumins, enzymes, phosphate and sulfate binding proteins,immunophilins, metallothionein, and various other receptor proteins. 6.The method of claim 1, wherein the second protein is an antigenicprotein or peptide.
 7. The method of claim 6, wherein the antigenicprotein or peptide is a protein or peptide derived from pathogenicbacterial, fungal or viral organisms, Streptococcus species, Candidaspecies, Brucella species, Salmonella species, Shigella species,Pseudomonas species, Bordetella species, Clostridium species, Norwalkvirus, Bacillus anthracis, Mycobacterium tuberculosis, humanimmunodeficiency virus (HIV), Chlamydia species, human Papillomaviruses,Influenza virus, Paramyxovirus species, Herpes virus, Cytomegalovirus,Varicella-Zoster virus, Epstein-Barr virus, Hepatitis viruses,Plasmodium species, Trichomonas species, sexually transmitted diseaseagents, viral encephalitis agents, protozoan disease agents, fungaldisease agents, bacterial disease agents, cancer cells, andcombinations.
 8. The method of claim 1, wherein the second protein is atherapeutic protein.
 9. The method of claim 1, wherein the secondprotein is an immunoregulatory protein.
 10. The method of claim 1,wherein a plurality of ClyA fusion proteins are displayed on the surfaceof a plurality of host cells.
 11. The method of claim 10, wherein eachof the plurality of ClyA fusion proteins comprises a different secondpolypeptide and wherein the plurality of ClyA fusion proteins form alibrary of polypeptides.
 12. The method of claim 1, wherein the ClyAprotein is a full length ClyA protein.
 13. The method of claim 1,wherein the second protein is fused to the C terminus of the ClyAprotein.
 14. The method of claim 1, wherein the second protein is fusedin the N terminus of the ClyA protein.
 15. A cell displaying a ClyAfusion protein, wherein said ClyA fusion protein comprises at least aportion of a ClyA protein and at least a portion of a second proteincoupled to said ClyA protein.
 16. A method of displaying a protein oncell vesicles, said method comprising: providing either a fusion proteincomprising at least a portion of a ClyA protein and at least a portionof a second protein coupled to said ClyA protein or a nucleic acidconstruct encoding the fusion protein and administering to a cell thefusion protein or the nucleic acid construct under conditions effectiveto display the fusion protein on the vesicles of the cell.
 17. Themethod of claim 16, wherein the second protein is a marker protein. 18.The method of claim 17, wherein the marker protein is a fluorescentprotein.
 19. The method of claim 16, wherein the second protein is aligand binding protein.
 20. The method of claim 19, wherein the ligandbinding protein is selected from the group consisting of high affinityantibody binding fragments, single-chain Fv antibody fragments,nanobodies, fluorobodies, aptamers, biotin-binding proteins,lipid-binding proteins, periplasmic binding proteins, lectins, serumalbumins, enzymes, phosphate and sulfate binding proteins,immunophilins, metallothionein, and various other receptor proteins. 21.The method of claim 16, wherein the second protein is an antigenicprotein or peptide.
 22. The method of claim 21, wherein the antigenicprotein or peptide is a protein or peptide derived from pathogenicbacterial, fungal or viral organisms, Streptococcus species, Candidaspecies, Brucella species, Salmonella species, Shigella species,Pseudomonas species, Bordetella species, Clostridium species, Norwalkvirus, Bacillus anthracis, Mycobacterium tuberculosis, humanimmunodeficiency virus (HIV), Chlamydia species, human Papillomaviruses,Influenza virus, Paramyxovirus species, Herpes virus, Cytomegalovirus,Varicella-Zoster virus, Epstein-Barr virus, Hepatitis viruses,Plasmodium species, Trichomonas species, sexually transmitted diseaseagents, viral encephalitis agents, protozoan disease agents, fungaldisease agents, bacterial disease agents, cancer cells, andcombinations.
 23. The method of claim 16, wherein the second protein isa therapeutic protein.
 24. The method of claim 16, wherein the secondprotein is an immunoregulatory protein.
 25. The method of claim 16,wherein a plurality of ClyA fusion proteins are displayed the surface ofa plurality of cell vesicles.
 26. The method of claim 25, wherein eachof the plurality of ClyA fusion proteins comprises a different secondpolypeptide and wherein the plurality of ClyA fusion proteins form alibrary of polypeptides.
 27. The method of claim 16, wherein the ClyAprotein is a full length ClyA protein.
 28. The method of claim 16,wherein the second protein is fused to the C terminus of the ClyAprotein.
 29. The method of claim 16, wherein the second protein is fusedto the N terminus of the ClyA protein.
 30. A vesicle displaying a ClyAfusion protein, wherein said fusion protein comprises at least a portionof a ClyA protein and at least a portion of a second protein coupled tosaid ClyA protein.
 31. A method of imaging cells, said methodcomprising: providing either a fusion protein comprising at least aportion of a ClyA protein and a marker protein coupled to said ClyAprotein or a nucleic acid construct encoding the fusion protein;administering to a cell the fusion protein or the nucleic acid constructunder conditions effective to display the fusion protein on the cell;and imaging the cell based on the presence of the marker protein. 32.The method of claim 31, wherein the marker protein is a fluorescentprotein.
 33. A method of sorting cells, said method comprising:providing either a fusion protein comprising at least a portion of aClyA protein and a marker protein coupled to said ClyA protein or anucleic acid construct encoding the fusion protein; administering to acell the fusion protein or the nucleic acid construct under conditionseffective to display the fusion protein on the cell; and sorting thecell based on the presence of the marker protein.
 34. The method ofclaim 33, wherein the marker protein is a fluorescent protein.
 35. Amethod of screening a library of candidate compounds to identify acompound that binds to a target protein, said method comprising:providing the library of candidate compounds to be screened; providing acell or a cell vesicle displaying a ClyA fusion protein comprising atleast a portion of a ClyA protein and at least a portion of a secondprotein, wherein the second protein of the ClyA fusion protein comprisesthe target protein; contacting the library of candidate compounds withthe cell or vesicle displaying the target protein under conditionseffective for the candidate compound to bind to the target protein; andidentifying compounds that bind to the target protein.
 36. A method ofdelivering a therapeutic agent to a cell, said method comprising:providing a vesicle displaying a ClyA fusion protein comprising at leasta portion of a ClyA protein and at least a portion of a second protein,wherein the vesicle contains the therapeutic agent to be delivered andwherein the second protein of the ClyA fusion protein comprises atargeting protein; and administering the vesicle to a cell underconditions effective to deliver the therapeutic agent to the cell. 37.The method of claim 36, wherein the therapeutic agent is a nucleic acid,protein, or small molecule.
 38. The method of claim 37, wherein thetherapeutic agent is an RNAi molecule.
 39. The method of claim 36,wherein the targeting protein comprises a ligand binding protein or anantigenic protein or peptide.
 40. A method of eliciting an immuneresponse in a mammal, said method comprising: providing a cell or a cellvesicle displaying a ClyA fusion protein comprising at least a portionof a ClyA protein and at least a portion of a second protein, whereinthe second protein of the ClyA fusion protein comprises an antigenicprotein or peptide capable of eliciting an immune response in themammal; and administering the cell or vesicle to the mammal underconditions effective to elicit the immune response.
 41. The methodaccording to claim 40 wherein the antigenic protein or peptide is aprotein or peptide derived from pathogenic bacterial, fungal or viralorganisms, Streptococcus species, Candida species, Brucella species,Salmonella species, Shigella species, Pseudomonas species, Bordetellaspecies, Clostridium species, Norwalk virus, Bacillus anthracis,Mycobacterium tuberculosis, human immunodeficiency virus (HIV),Chlamydia species, human Papillomaviruses, Influenza virus,Paramyxovirus species, Herpes virus, Cytomegalovirus, Varicella-Zostervirus, Epstein-Barr virus, Hepatitis viruses, Plasmodium species,Trichomonas species, sexually transmitted disease agents, viralencephalitis agents, protozoan disease agents, fungal disease agents,bacterial disease agents, cancer cells, and combinations.
 42. A drug orvaccine delivery vehicle comprising a vesicle displaying a ClyA fusionprotein comprising at least a portion of a ClyA protein and at least aportion of a second protein.
 43. The delivery vehicle of claim 42,wherein said vesicle contains the drug or vaccine to be delivered andwherein the second protein of the ClyA fusion protein comprises atargeting protein.
 44. The delivery vehicle of claim 43, wherein saidtargeting protein is a cell specific ligand binding protein or anantigenic protein or peptide.
 45. The delivery vehicle of claim 43,wherein the vesicle contains a nucleic acid, protein, or small moleculeto be delivered.
 46. The delivery vehicle of claim 45, wherein thevesicle contains an RNAi molecule.
 47. The delivery vehicle of claim 42,wherein the second protein of the ClyA fusion protein displayed on thevesicle is an antigenic protein or peptide.
 48. The delivery vehicle ofclaim 47, wherein the second protein of the ClyA fusion proteindisplayed on the vesicle surface is a vaccine subunit protein.